Anti-amyloid antibodies

ABSTRACT

Methods useful for effecting prophylaxis or treatment of amyloidosis, including AA Amyloidosis and AL amyloidosis, by administering peptides comprising neoepitopes, such as AA fragments from a C-terminal region of AA, and antibodies specific for neoepitopes of aggregated amyloid proteins, for example, antibodies specific for the C-terminal region of AA fibrils. Antibodies for inhibition of formation and/or increasing clearance of amyloid deposits in a patient thus effecting prophylaxis or treating amyloid disease.

CROSS-REFERENCES TO RELATED APPLICATIONS

This is a continuation of pending U.S. application Ser. No. 13/032,412,filed Feb. 22, 2011, which is a continuation of U.S. application Ser.No. 12/345,650, filed Dec. 29, 2008, which issued as U.S. Pat. No.7,928,203, and which claims priority to U.S. Provisional Application No.61/095,932, filed Sep. 10, 2008, and to U.S. Provisional Application No.61/007,544, filed Dec. 28, 2007, each of which is incorporated herein byreference in its entirety.

TECHNICAL FIELD

The invention resides in the technical fields of immunology andmedicine.

BACKGROUND OF THE INVENTION

Amyloidosis is a general term that describes a number of diseasescharacterized by the existence of pathological forms of amyloidproteins, often involving extracellular deposition of protein fibrils,which form numerous “amyloid deposits” or “amyloid plaques,” which mayoccur in local sites or systematically. These deposits or plaques arecomposed primarily of a naturally occurring soluble protein or peptide,assembled into extensive insoluble deposits 10-100 μm in diameter in avariety of tissue sites. The deposits are composed of generally lateralaggregates of fibrils that are approximately 10-15 nm in diameter.Amyloid fibrils produce a characteristic apple green birefringence inpolarized light, when stained with Congo Red dye. Generally, thefibrillar composition of these deposits is an identifying characteristicfor the various forms of amyloid disease.

The peptides or proteins forming the plaque deposits are often producedfrom a larger precursor protein. More specifically, the pathogenesis ofamyloid aggregates such as fibril deposits generally involvesproteolytic cleavage of an “abnormal” precursor protein into fragmentsthat aggregate into anti-parallel β pleated sheets.

The fibrillar composition of these deposits is an identifyingcharacteristic for the various forms of amyloid disease. For example,intracerebral and cerebrovascular deposits composed primarily of fibrilsof beta amyloid peptide (β-AP) are characteristic of Alzheimer's disease(both familial and sporadic forms), islet amyloid protein peptide (IAPP;amylin) is characteristic of the fibrils in pancreatic islet cellamyloid deposits associated with type II diabetes, and β2-microglobulinis a major component of amyloid deposits which form as a consequence oflong term hemodialysis treatment. More recently, prion-associateddiseases, such as Creutzfeld-Jacob disease, have also been recognized asamyloid diseases.

In general, primary amyloidoses of the disease are characterized by thepresence of “amyloid light chain-type” (AL-type) protein fibrils, sonamed for the homology of the N-terminal region of the AL fibrils to thevariable fragment of immunoglobulin light chain (kappa or lambda).

The various forms of disease have been divided into classes, mostly onthe basis of whether the amyloidosis is associated with an underlyingsystematic illness. Thus, certain disorders are considered to be primaryamyloidoses, in which there is no evidence for preexisting or coexistingdisease. In secondary or reactive (AA type) amyloidosis characterized bythe presence deposition of amyloid protein A (AA) fibrils, there is anunderlying or associated chronic inflammatory or infectious diseasestate.

Heredofamilial amyloidoses may have associated neuropathic, renal, orcardiovascular deposits of the ATTR transthyretin type. Otherheredofamilial amyloidoses include other syndromes and may havedifferent amyloid components (e.g., familial Mediterranean fever whichis characterized by AA fibrils). Other forms of amyloidosis includelocal forms, characterized by focal, often tumor-like deposits thatoccur in isolated organs. Other amyloidoses are associated with aging,and are commonly characterized by plaque formation in the heart orbrain. Also common are amyloid deposits associated with long termhemodialysis. These and other forms of amyloid disease are summarized inTable 1 (Tan, S. Y. and Pepys, Histopathology 25:403-414, 1994;Harrison's Handbook of Internal Medicine, 13^(th) Ed., Isselbacher, K.J., et al, eds, McGraw-Hill, San Francisco, 1995) and are described inU.S. Pat. Nos. 6,875,434, 6,890,535, 6,913,745, 6,923,964, and6,936,246, which are incorporated by reference herein in their entirety.

TABLE 1 Classification of Amyloid Diseases Amyloid Protein/ ProteinPeptide Precursor Protein Variants Clinical AA Serum Amyloid A Reactive(secondary) Protein (ApoSSA) Amyloidosis: Familial Mediterranean feverFamilial amyloid nephropathy with urticaria and deafness (Muckle- Wellssyndrome) AA Serum amyloid A Reactive systemic protein amyloidosisassociated (ApoSSA) with systemic inflammatory diseases AL MonoclonalAk, A, (e.g., AkIII) Idiopathic (primary) immunoglobulin lightAmyloidosis: myeloma or chains (kappa, lambda) macroglobulinemia-associated; systemic amyloidosis associated with immunocyte dyscrasia;monoclonal gammopathy; occult dyscrasia; local nodular amyloidosisassociated with chronic inflammatory diseases AH IgG (1(γ1)) Aγ1 Heavychain amyloidosis associated with several immunocyte dyscrasias ATTRTransthyretin (TTR) At least 30 Familial amyloid known pointpolyneuropathy mutations (e.g., Met 30, Portuguese) ATTR Transthyretin(TTR) e.g., Met 111 Familial amyloid cardiomyopathy (Danish) ATTRTransthyretin (TTR) Wild-type TTR Systemic senile or Ile 122 amyloidosisAapoAI ApoAI Arg 26 Familial amyloid polyneuropathy Agel Gelsolin Asn187 Familial amyloidosis (Finnish) Acys Cystatin C Gln 68 Hereditarycerebral hemorrhage with amyloidosis (Icelandic) Aβ Amyloid β proteinVarious: Gln 618, Alzheimer's disease precursor (e.g. (β- Down'ssyndrome APP₆₉₅) Hereditary cerebral hemorrhage amyloidosis (Dutch)Sporadic cerebral amyloid angiopathy Inclusion body myositis AB₂M Beta₂microglobulin Associated with chronic hemodialysis Acal (Pro)calcitonin(Pro)calcitonin Medullary carcinoma of thyroid Focal Senile Amyloidoses:AANF Atrial natriuretic factor Isolated atrial amyloid Aβ β-amyloidprecursor Brain protein SVEP^(a) — Seminal vesicles AB₂M Beta₂microglobulin Prostate Keratin Primary localized cutaneous amyloid(macular, papular) PrP Prion precursor protein Scrapie protein SporadicCreutzfeldt-Jacob (33-35 kDa cellular 27-30 kDa Disease form) Kuru(transmissible spongiform encephalopathies, prion diseases) AIAPP Isletamyloid Islets of Langerhans polypeptide (IAPP) Diabetes type II,Insulinoma Peptide e.g., precalcitonin Exocrine amyloidosis, hormones,associated with fragments APUDomas ^(a)Seminal vesicle exocrine protein

Often, fibrils forming the bulk of an amyloid deposit are derived fromone or more primary precursor proteins or peptides, and are usuallyassociated with sulfated glycosaminoglycans. In addition, amyloiddeposits may include minor proteins and peptides of various types, alongwith other components, such as proteoglycans, gangliosides and othersugars, as described in more detail in the sections that follow.

AA fibrils are composed of peptide fragments that range in size but aregenerally about 8000 daltons (AA peptide or protein) formed byproteolytic cleavage of serum amyloid A protein (SSA), a circulatingapolipoprotein which is present in HDL particles and which issynthesized in hepatocytes in response to such cytokines as interleukin(IL)-1 and IL-6, as well as tumor necrosis factor α. See Husby, G. etal. Amyloid 1, 119-137 (1994). The proteolytic cleavage results in thepathologic deposition of an ˜76-residue N-terminal two thirds of the SAAprotein. In humans, the plasma concentration of SAA normally is ˜0.1mg/ml but can increase over 1,000-fold in response to an inflammatorystimulus. As part of this process, the SAA molecule undergoesproteolysis and the N-terminal cleavage product is depositedsystemically as AA fibrils in vital organs, including the liver, spleen,kidneys, and adrenal glands. Deposition is also common in the heart andgastrointestinal tract.

Generally, AA amyloidosis is a manifestation of diseases that provoke asustained acute phase response. Such diseases include chronicinflammatory disorders, chronic local or systemic microbial infections,and malignant neoplasms. AA amyloid diseases include, but are notlimited to inflammatory diseases, such as rheumatoid arthritis, juvenilechronic arthritis, ankylosing spondylitis, psoriasis, psoriaticarthropathy, Reiter's syndrome, Adult Still's disease, Behcet'ssyndrome, and Crohn's disease. AA deposits are also produced as a resultof chronic microbial infections, such as leprosy, tuberculosis,bronchiectasis, decubitus ulcers, chronic pyelonephritis, osteomyelitis,and Whipple's disease. Certain malignant neoplasms can also result in AAfibril amyloid deposits. These include such conditions such as Hodgkin'slymphoma, renal carcinoma, carcinomas of gut, lung and urogenital tract,basal cell carcinoma, and hairy cell leukemia. AA amyloid disease mayalso result from inherited inflammatory diseases such as FamilialMediterranean Fever. Additionally, AA amyloid disease may result fromlymphoproliferative disorders such as Castleman's Disease.

AA Amyloidosis is insidious and progressive. Symptoms are generallypresented in later stages of the disease. Frequently the patient isundiagnosed until significant organ damage has occurred. AA fibrils aredeposited in vital organs leading to organ dysfunction and subsequentlyto death. The five year survival rate is 45-50%. Median survival afterdiagnosis is 4-8 years. End stage Renal Disease is the cause of death in40-60% of cases. See Gillmore J. D. et al., Lancet 358:24-9 (2001).

Currently, there are no approved specific, amyloid-directed treatmentsfor any of the amyloid diseases, including AA Amyloidosis. See GillmoreJ. D. et al., Lancet 358:24-9 (2001). Where there is an underlying orassociated disease state, therapy directed towards decreasing theproduction of amyloidogenic protein by treating the underlying disease.For example, current treatment strategy for AA Amyloidosis is to targetunderlying inflammation, reducing ApoSSA levels to below 10 mg/l.Currently employed therapies include chemotherapy (cholorambucil andMTX), immuno-suppressants (azathioprine), anti-inflammatory drugs(colchicine) and TNF inhibitors. The invention thus fulfills alongstanding need for therapeutic regimes for preventing or amelioratingthe effects of AA Amyloidosis.

SUMMARY OF THE INVENTION

The present invention provides an isolated human, humanized, or chimericantibody, or antigen-binding fragment thereof, that specifically bindsto an epitope within residues 70-76 of human amyloid A peptide, forexample, an epitope within residues 70-76 of SEQ ID NO: 2 or an epitopecomprising residues set forth as SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, or11. Antibodies or antigen-binding fragments of the invention includethose that compete for binding to human amyloid A peptide with antibody2A4 produced by ATCC Accession Number PTA-9662 or with antibody 7D8produced by ATCC Accession Number PTA-9468. Additional antibodies of theinvention compete for binding to human amyloid A peptide with anantibody having a light chain variable region set forth as residues20-131 of SEQ ID NO: 152 or residues 20-131 of 153 and a heavy chainvariable region set forth as residues 20-138 of SEQ ID NO: 154.

The disclosed antibodies include humanized and chimeric versions ofantibody 2A4 produced by ATCC Accession Number PTA-9662 or a humanizedor chimeric version of antibody 7D8 produced by ATCC Accession NumberPTA-9468.

For example, representative antibodies and antigen-binding fragmentscomprise a light chain variable region comprising one or morecomplementarity regions of a 2A4 light chain variable region set forthas residues 20-131 of SEQ ID NO: 152 or one or more complementarityregions of a 7D8 light chain variable region set forth as residues20-131 of SEQ ID NO: 153. As another example, representative antibodiesand antigen-binding fragments comprise a light chain variable regioncomprising two complementarity regions of a 2A4 light chain variableregion set forth as residues 20-131 of SEQ ID NO: 152 or twocomplementarity regions of a 7D8 light chain variable region set forthas residues 20-131 of SEQ ID NO: 153. Additional representativeantibodies and antigen-binding fragments comprise a light chain variableregion comprising three complementarity regions of a 2A4 light chainvariable region set forth as residues 20-131 of SEQ ID NO: 152 or threecomplementarity regions of a 7D8 light chain variable region set forthas residues 20-131 of SEQ ID NO: 153. Representative humanized versionsof a 2A4 or 7D8 antibody comprise at least one light chain frameworkresidue selected from the group consisting of L87 and L90 (Kabatnumbering convention), which is occupied by Y and F, respectively, andwherein the remainder of the light chain variable region is occupied bya corresponding residue in a human acceptor immunoglobulin light chainvariable region. Representative antibodies and antigen-binding fragmentscomprise at least one light chain framework residue selected from thegroup consisting of +7, +14, +15, +17, +18, +50, +75, +88, +92, and +109(linear numbering), which is occupied by T, S, L, D, Q, K, Y, L, F, andL, respectively, and wherein the remainder of the light chain variableregion is occupied by a corresponding residue in a human acceptorimmunoglobulin light chain variable region. For example, representativeantibodies and antigen-binding fragments comprise at least one lightchain framework residue selected from the group consisting of +75 and+92 (linear numbering), which is occupied by Y and F, respectively, andwherein the remainder of the light chain variable region is occupied bya corresponding residue in a human acceptor immunoglobulin light chainvariable region. In other representative antibodies and antigen-bindingfragments of the invention, the light chain variable region comprises aframework residue at +105 (linear numbering) occupied by Q.

For example, antibodies and antigen-binding fragments of the inventioninclude those comprising a light chain variable region comprising aframework residue at +7 (linear numbering) occupied by T, wherein theremainder of the light chain variable region is occupied by acorresponding residue in a human acceptor immunoglobulin light chainvariable region; antibodies and antigen-binding fragments a light chainvariable region comprising a framework residue at +14 (linear numbering)occupied by S, wherein the remainder of the light chain variable regionis occupied by a corresponding residue in a human acceptorimmunoglobulin light chain variable region; antibodies andantigen-binding fragments a light chain variable region comprising aframework residue at +15 (linear numbering) occupied by L, wherein theremainder of the light chain variable region is occupied by acorresponding residue in a human acceptor immunoglobulin light chainvariable region; antibodies and antigen-binding fragments a light chainvariable region comprising a framework residue at +17 (linear numbering)occupied by D, wherein the remainder of the light chain variable regionis occupied by a corresponding residue in a human acceptorimmunoglobulin light chain variable region; antibodies andantigen-binding fragments a light chain variable region comprising aframework residue at +18 (linear numbering) occupied by Q, wherein theremainder of the light chain variable region is occupied by acorresponding residue in a human acceptor immunoglobulin light chainvariable region; antibodies and antigen-binding fragments a light chainvariable region comprising a framework residue at +50 (linear numbering)occupied by K, wherein the remainder of the light chain variable regionis occupied by a corresponding residue in a human acceptorimmunoglobulin light chain variable region; antibodies andantigen-binding fragments a light chain variable region comprising aframework residue at +75 (linear numbering) occupied by Y, wherein theremainder of the light chain variable region is occupied by acorresponding residue in a human acceptor immunoglobulin light chainvariable region; antibodies and antigen-binding fragments a light chainvariable region comprising a framework residue at +88 (linear numbering)occupied by L, wherein the remainder of the light chain variable regionis occupied by a corresponding residue in a human acceptorimmunoglobulin light chain variable region; antibodies andantigen-binding fragments a light chain variable region comprising aframework residue at +92 (linear numbering) occupied by F, wherein theremainder of the light chain variable region is occupied by acorresponding residue in a human acceptor immunoglobulin light chainvariable region; antibodies and antigen-binding fragments a light chainvariable region comprising a framework residue at +109 (linearnumbering) occupied by L, wherein the remainder of the light chainvariable region is occupied by a corresponding residue in a humanacceptor immunoglobulin light chain variable region; and antibodies andantigen-binding fragments a light chain variable region comprising aframework residue at +105 (linear numbering) occupied by Q, wherein theremainder of the light chain variable region is occupied by acorresponding residue in a human acceptor immunoglobulin light chainvariable region.

Human acceptor immunoglobulin light chain variable regions used in theinvention include human kappa subgroup 2 light chain variable region(Kabat convention), for example, human subgroup 2 light chain variableregion from human germline VKIIA19/A3, such as human Vk light chainvariable region comprising a sequence set forth as SEQ ID NO: 166 or167. In particular aspects of the invention, antibodies andantigen-binding fragments comprise a light chain variable regioncomprising an amino acid sequence set forth as residues 20-131 of SEQ IDNO: 152, residues 20-131 of SEQ ID NO: 153, or set forth as SEQ ID NO:155, 156, 157, 158, 159, 160, 174, 175, or 176.

Representative antibodies and antigen-binding fragments of the inventionalso include those comprising a heavy chain variable region comprisingone or more complementarity regions of a 2A4 heavy chain variable regionset forth as residues 20-138 of SEQ ID NO: 154, for example, a heavychain variable region comprising two complementarity regions of a 2A4heavy chain variable region set forth as residues 20-138 of SEQ ID NO:154, or a heavy chain variable region comprising three complementarityregions of a 2A4 heavy chain variable region set forth as residues20-138 of SEQ ID NO: 154. Representative humanized 2A4 and 7D8antibodies and antigen-binding fragments comprise at least one heavychain framework residue selected from the group consisting of H37, H49,H70, and H93 (Kabat numbering convention), which is occupied by I, A, F,or V, respectively, and wherein the remainder of the heavy chainvariable region is occupied by a corresponding residue in a humanacceptor immunoglobulin heavy chain variable region. Representativehumanized antibodies and antigen-binding fragments comprise at least oneheavy chain framework residue selected from the group consisting of +10,+15, +19, +37, +49, +73, +78, +79, +80, +87, +95, +99, +119 (linearnumbering), which is occupied by R, K, K, I, A, F, Q, S, M, N, M, V, orA, respectively, and wherein the remainder of the heavy chain variableregion is occupied by a corresponding residue in a human acceptorimmunoglobulin heavy chain variable region. For example, representativehumanized antibodies and antigen-binding fragments comprise at least oneheavy chain framework residue selected from the group consisting of +37,+49, +73, and +99 (linear numbering), which is occupied by I, A, F, orV, respectively, and wherein the remainder of the heavy chain variableregion is occupied by a corresponding residue in a human acceptorimmunoglobulin heavy chain variable region.

For example, antibodies and antigen-binding fragments of the inventioninclude those comprising a heavy chain variable region comprising aframework residue at +10 (linear numbering) occupied by R, wherein theremainder of the heavy chain variable region is occupied by acorresponding residue in a human acceptor immunoglobulin heavy chainvariable region; antibodies and antigen-binding fragments a heavy chainvariable region comprising a framework residue at +15 (linear numbering)occupied by K, wherein the remainder of the heavy chain variable regionis occupied by a corresponding residue in a human acceptorimmunoglobulin heavy chain variable region; antibodies andantigen-binding fragments a heavy chain variable region comprising aframework residue at +19 (linear numbering) occupied by K, wherein theremainder of the heavy chain variable region is occupied by acorresponding residue in a human acceptor immunoglobulin heavy chainvariable region; antibodies and antigen-binding fragments a heavy chainvariable region comprising a framework residue at +37 (linear numbering)occupied by I, wherein the remainder of the heavy chain variable regionis occupied by a corresponding residue in a human acceptorimmunoglobulin heavy chain variable region; antibodies andantigen-binding fragments a heavy chain variable region comprising aframework residue at +49 (linear numbering) occupied by A, wherein theremainder of the heavy chain variable region is occupied by acorresponding residue in a human acceptor immunoglobulin heavy chainvariable region; antibodies and antigen-binding fragments a heavy chainvariable region comprising a framework residue at +73 (linear numbering)occupied by F, wherein the remainder of the heavy chain variable regionis occupied by a corresponding residue in a human acceptorimmunoglobulin heavy chain variable region; antibodies andantigen-binding fragments a heavy chain variable region comprising aframework residue at +78 (linear numbering) occupied by Q, wherein theremainder of the heavy chain variable region is occupied by acorresponding residue in a human acceptor immunoglobulin heavy chainvariable region; antibodies and antigen-binding fragments a heavy chainvariable region comprising a framework residue at +79 (linear numbering)occupied by S, wherein the remainder of the heavy chain variable regionis occupied by a corresponding residue in a human acceptorimmunoglobulin heavy chain variable region; antibodies andantigen-binding fragments a heavy chain variable region comprising aframework residue at +80 (linear numbering) occupied by M, wherein theremainder of the heavy chain variable region is occupied by acorresponding residue in a human acceptor immunoglobulin heavy chainvariable region; antibodies and antigen-binding fragments a heavy chainvariable region comprising a framework residue at +87 (linear numbering)occupied by N, wherein the remainder of the heavy chain variable regionis occupied by a corresponding residue in a human acceptorimmunoglobulin heavy chain variable region; antibodies andantigen-binding fragments a heavy chain variable region comprising aframework residue at +95 (linear numbering) occupied by M, wherein theremainder of the heavy chain variable region is occupied by acorresponding residue in a human acceptor immunoglobulin heavy chainvariable region; antibodies and antigen-binding fragments a heavy chainvariable region comprising a framework residue at +99 (linear numbering)occupied by V, wherein the remainder of the heavy chain variable regionis occupied by a corresponding residue in a human acceptorimmunoglobulin heavy chain variable region; and antibodies andantigen-binding fragments a heavy chain variable region comprising aframework residue at +109 (linear numbering) occupied by A, wherein theremainder of the heavy chain variable region is occupied by acorresponding residue in a human acceptor immunoglobulin heavy chainvariable region.

Human acceptor immunoglobulin heavy chain variable regions include ahuman gamma subgroup 3 heavy chain variable region (Kabat convention),for example, human gamma subgroup 3 heavy chain variable regioncomprising a sequence set forth as SEQ ID NO: 165, such as a heavy chainvariable region comprising an amino acid sequence set forth as residues20-138 of SEQ ID NO: 154 or set forth as SEQ ID NO: 161, 162, or 163.

Additional representative antibodies and antigen-binding fragmentscomprise a light chain variable region comprising three complementaritydetermining regions of a 2A4 light chain variable region set forth asresidues 20-131 of SEQ ID NO: 152 or three complementarity regions of a7D8 light chain variable region set forth as residues 20-131 of SEQ IDNO: 153, and a heavy chain variable region comprising threecomplementarity regions of a 2A4 heavy chain variable region set forthas residues 20-138 of SEQ ID NO: 154. For example, such antibodies andantigen-binding fragments include those having a light chain variableregion comprising three complementarity determining regions set forth asSEQ ID NOs: 168, 169, and 170, and a heavy chain variable regioncomprising three complementarity regions set forth as SEQ ID NOs: 171,172, and 173. As another example, such antibodies and antigen-bindingfragments include those having a light chain variable region comprisingthree complementarity determining regions set forth as SEQ ID NOs: 177,169, and 170, and a heavy chain variable region comprising threecomplementarity regions set forth as SEQ ID NOs: 171, 172, and 173. Asanother example, such antibodies and antigen-binding fragments includethose comprising a light chain variable region comprising an amino acidsequence set forth as residues 20-131 of SEQ ID NO: 152 or as residues20-131 of SEQ ID NO: 153, and a heavy chain variable region comprisingan amino acid sequence set forth as residues 20-138 of SEQ ID NO: 154.As another example, such antibodies and antigen-binding fragmentsinclude those having a light chain variable region comprising an aminoacid sequence set forth as SEQ ID NO: 155, 156, 157, 158, 159, 160, 174,175, or 176, and a heavy chain variable region comprising an amino acidsequence set forth as SEQ ID NO: 161, 162, or 163.

In particular aspects of the invention, an antibody or antigen-bindingfragment comprises a light chain variable region comprising an aminoacid sequence set forth as SEQ ID NO: 155, and a heavy chain variableregion comprising an amino acid sequence set forth as SEQ ID NO: 161; alight chain variable region comprising an amino acid sequence set forthas SEQ ID NO: 155, and a heavy chain variable region comprising an aminoacid sequence set forth as SEQ ID NO: 162; a light chain variable regioncomprising an amino acid sequence set forth as SEQ ID NO: 155, and aheavy chain variable region comprising an amino acid sequence set forthas SEQ ID NO: 163; a light chain variable region comprising an aminoacid sequence set forth as SEQ ID NO: 156, and a heavy chain variableregion comprising an amino acid sequence set forth as SEQ ID NO: 161; alight chain variable region comprising an amino acid sequence set forthas SEQ ID NO: 156, and a heavy chain variable region comprising an aminoacid sequence set forth as SEQ ID NO: 162; a light chain variable regioncomprising an amino acid sequence set forth as SEQ ID NO: 156, and aheavy chain variable region comprising an amino acid sequence set forthas SEQ ID NO: 163; a light chain variable region comprising an aminoacid sequence set forth as SEQ ID NO: 157, and a heavy chain variableregion comprising an amino acid sequence set forth as SEQ ID NO: 161; alight chain variable region comprising an amino acid sequence set forthas SEQ ID NO: 157, and a heavy chain variable region comprising an aminoacid sequence set forth as SEQ ID NO: 162; a light chain variable regioncomprising an amino acid sequence set forth as SEQ ID NO: 157, and aheavy chain variable region comprising an amino acid sequence set forthas SEQ ID NO: 163; a light chain variable region comprising an aminoacid sequence set forth as SEQ ID NO: 158, and a heavy chain variableregion comprising an amino acid sequence set forth as SEQ ID NO: 161; alight chain variable region comprising an amino acid sequence set forthas SEQ ID NO: 158, and a heavy chain variable region comprising an aminoacid sequence set forth as SEQ ID NO: 162; a light chain variable regioncomprising an amino acid sequence set forth as SEQ ID NO: 158, and aheavy chain variable region comprising an amino acid sequence set forthas SEQ ID NO: 163; a light chain variable region comprising an aminoacid sequence set forth as SEQ ID NO: 159, and a heavy chain variableregion comprising an amino acid sequence set forth as SEQ ID NO: 161; alight chain variable region comprising an amino acid sequence set forthas SEQ ID NO: 159, and a heavy chain variable region comprising an aminoacid sequence set forth as SEQ ID NO: 162; a light chain variable regioncomprising an amino acid sequence set forth as SEQ ID NO: 159, and aheavy chain variable region comprising an amino acid sequence set forthas SEQ ID NO: 163; a light chain variable region comprising an aminoacid sequence set forth as SEQ ID NO: 160, and a heavy chain variableregion comprising an amino acid sequence set forth as SEQ ID NO: 161; alight chain variable region comprising an amino acid sequence set forthas SEQ ID NO: 160, and a heavy chain variable region comprising an aminoacid sequence set forth as SEQ ID NO: 162; a light chain variable regioncomprising an amino acid sequence set forth as SEQ ID NO: 160, and aheavy chain variable region comprising an amino acid sequence set forthas SEQ ID NO: 163; SEQ ID NO: 174, and a heavy chain variable regioncomprising an amino acid sequence set forth as SEQ ID NO: 161; a lightchain variable region comprising an amino acid sequence set forth as SEQID NO: 174, and a heavy chain variable region comprising an amino acidsequence set forth as SEQ ID NO: 162; a light chain variable regioncomprising an amino acid sequence set forth as SEQ ID NO: 174, and aheavy chain variable region comprising an amino acid sequence set forthas SEQ ID NO: 163; a light chain variable region comprising an aminoacid sequence set forth as SEQ ID NO: 175, and a heavy chain variableregion comprising an amino acid sequence set forth as SEQ ID NO: 161; alight chain variable region comprising an amino acid sequence set forthas SEQ ID NO: 175, and a heavy chain variable region comprising an aminoacid sequence set forth as SEQ ID NO: 162; a light chain variable regioncomprising an amino acid sequence set forth as SEQ ID NO: 175, and aheavy chain variable region comprising an amino acid sequence set forthas SEQ ID NO: 163; a light chain variable region comprising an aminoacid sequence set forth as SEQ ID NO: 176, and a heavy chain variableregion comprising an amino acid sequence set forth as SEQ ID NO: 161; alight chain variable region comprising an amino acid sequence set forthas SEQ ID NO: 176, and a heavy chain variable region comprising an aminoacid sequence set forth as SEQ ID NO: 162; or a light chain variableregion comprising an amino acid sequence set forth as SEQ ID NO: 176,and a heavy chain variable region comprising an amino acid sequence setforth as SEQ ID NO: 163.

Also provided are isolated nucleic acids encoding a human, humanized, orchimeric antibody, or antigen-binding fragment thereof, thatspecifically binds to an epitope within residues 70-76 of human amyloidA peptide, including all such antibodies and antigen-binding fragmentsas described herein above and as set forth in the claims. Furtherprovided are cells expressing such nucleic acids.

In other aspects, the present invention provides an isolated antibody,or antigen-binding fragment thereof, which specifically binds to anepitope comprising X₁EDX₂ in an aggregated amyloid protein, wherein X₁and X₂ are any amino acid. Such antibodies and antigen-binding fragmentsinclude human, humanized, or chimeric antibodies, and antigen-bindingfragments thereof, for example, those that specifically bind to anepitope within residues 70-76 of human amyloid A peptide.

Additional representative antibodies and antigen-binding fragmentsinclude those wherein X₁ is H, T, F, S, P, A, L, C, Q, R, E, K, D, G, V,Y, I, or W, and wherein X₂ is T, S, E, R, I, V, F, D, A, G, M, L, N, P,C, K, Y, or Q; or X₁ is H, T, F, S, P, or A and wherein X₂ is T, S, E,R, I, V, F, D, or A; or X₁ is H, T, F, or A; or X₂ is T, S, E, D, or A;or X₁ is H, T, F, or A and X₂ is T, S, E, D, or A; or X₁ is H, T, or Aand X₂ is T, S, E, or A; or X₁ is H or A and X₂ is T, S, or A; or X₁ isH and X₂ is T or A; or X₁ is A and X₂ is S, T, E or V; or X₁ is A and X₂is S, T or E; or X₁ is T and X₂ is E; or X₁ is F and X₂ is D; or X₁ is Sand X₂ is E, F or A; or X₁ is P and X₂ is E, I or F. For example, suchantibodies and antigen-binding fragments bind an epitope consisting ofan amino acid sequence selected from the group consisting of GHEDT (SEQID NO: 3), HEDT (SEQ ID NO: 12), AEDS (SEQ ID NO: 13), AEDT (SEQ ID NO:14), HEDA (SEQ ID NO: 15), TEDE (SEQ ID NO: 16), FEDD (SEQ ID NO: 17),SEDE (SEQ ID NO: 18), AEDE (SEQ ID NO: 19), PEDE (SEQ ID NO: 20), PEDI(SEQ ID NO: 21), PEDF (SEQ ID NO: 22), AEDV (SEQ ID NO: 23), SEDF (SEQID NO: 24), and SEDA (SEQ ID NO: 25); or an epitope consisting of anamino acid sequence selected from the group consisting of GHEDT (SEQ IDNO: 3), HEDT (SEQ ID NO: 12), AEDS (SEQ ID NO: 13), AEDT (SEQ ID NO:14), HEDA (SEQ ID NO: 15), TEDE (SEQ ID NO: 16), FEDD (SEQ ID NO: 17),SEDE (SEQ ID NO: 18), AEDE (SEQ ID NO: 19), PEDE (SEQ ID NO: 20), PEDI(SEQ ID NO: 21), PEDF (SEQ ID NO: 22), SEDF (SEQ ID NO: 24), and SEDA(SEQ ID NO: 25); or an epitope consisting of an amino acid sequenceselected from the group consisting of GHEDT (SEQ ID NO: 3), HEDT (SEQ IDNO: 12), AEDS (SEQ ID NO: 13), AEDT (SEQ ID NO: 14), HEDA (SEQ ID NO:15), and TEDE (SEQ ID NO: 16). The disclosed epitopes may be found in anaggregated amyloid protein, for example, an epitope comprising an aminoacid sequence selected from the group consisting of GHGAEDS (SEQ ID NO:4), GHDAEDS (SEQ ID NO: 5), GDHAEDS (SEQ ID NO: 7), STVIEDS (SEQ ID NO:8), and GRGHEDT (SEQ ID NO: 9); or an epitope comprising an amino acidsequence GHGAEDS (SEQ ID NO:4); or an epitope comprising amino acidsHEDT (SEQ ID NO: 12); or an epitope comprising amino acids HEDA (SEQ IDNO: 15); or an epitope comprising amino acids AEDS (SEQ ID NO: 13) or anepitope comprising amino acids AEDT (SEQ ID NO: 14); or an epitopecomprising amino acids TEDE (SEQ ID NO: 16); or an epitope comprisingthe amino acid sequence AEDV (SEQ ID NO: 23); or an epitope comprisingthe amino acid sequence SEDF (SEQ ID NO: 24) or PEDF (SEQ ID NO: 22); oran epitope of comprising an amino sequence selected from the groupconsisting of PEDS (SEQ ID NO: 26), PEDL (SEQ ID NO: 27), TEDV (SEQ IDNO: 28), AEDE (SEQ ID NO: 19), SEDI (SEQ ID NO: 29) and TEDT (SEQ ID NO:30); or an epitope comprising an amino sequence selected from the groupconsisting of LEDG (SEQ ID NO: 31), AEDM (SEQ ID NO: 32), HEDS (SEQ IDNO: 33), CEDD (SEQ ID NO: 34), QEDS (SEQ ID NO: 35), REDS (SEQ ID NO:36), TEDG (SEQ ID NO: 16), QEDR (SEQ ID NO: 38), TEDL (SEQ ID NO: 39),PEDN (SEQ ID NO: 40), EEDP (SEQ ID NO: 41), LEDL (SEQ ID NO: 42), KEDA(SEQ ID NO: 43), SEDC (SEQ ID NO: 44), EEDD (SEQ ID NO: 45), SEDK (SEQID NO: 46), DEDD (SEQ ID NO: 47), DEDG (SEQ ID NO: 13), LEDE (SEQ ID NO:49), GEDA (SEQ ID NO: 13), VEDF (SEQ ID NO: 51), YEDE (SEQ ID NO: 52),IEDL (SEQ ID NO: 53), WEDY (SEQ ID NO: 54), DEDW (SEQ ID NO: 55), SEDL(SEQ ID NO: 56), YEDQ (SEQ ID NO: 57), LEDW (SEQ ID NO: 58), YEDR (SEQID NO: 59) and PEDK (SEQ ID NO: 60).

The antibodies and antigen-binding fragments described herein includethose that bind to the amyloid protein in monomeric form with anaffinity of less than about 10⁷ M⁻¹. Representative amyloid proteinsinclude serum amyloid A protein (SAA), immunoglobulin light chainprotein (such as Vλ6 Wil and Vκ), human islet amyloid precursorpolypeptide (IAPP), beta amyloid peptide, transthyretin (TTR), andApoA1.

Also provided are isolated nucleic acids encoding an antibody, orantigen-binding fragment thereof, which specifically binds to an epitopecomprising X₁EDX₂ in an aggregated amyloid protein, wherein X₁ and X₂are any amino acid, including all such antibodies and antigen-bindingfragments as described herein above and as set forth in the claims.Further provided are cells expressing such nucleic acids.

The present invention further provides methods of therapeuticallytreating or prophylactically treating a subject having AA amyloidosisusing a human, humanized, or chimeric antibody, or antigen-bindingfragment thereof, that specifically binds to an epitope within residues70-76 of human amyloid A peptide, for example, an epitope withinresidues 70-76 of SEQ ID NO: 2. Subjects that may benefit from thedisclosed therapeutic methods of treating AA amyloidosis include thosesubjects suffering from an amyloid disease selected from the groupconsisting of rheumatoid arthritis, juvenile chronic arthritis,ankylosing spondylitis, psoriasis, psoriatic arthropathy, Reiter'ssyndrome, Adult Still's disease, Behcet's syndrome, Crohn's disease,leprosy, tuberculosis, bronchiectasis, decubitus ulcers, chronicpyelonephritis, osteomyelitis, Whipple's disease, Hodgkin's lymphoma,renal carcinoma, carcinomas of gut, lung and urogenital tract, basalcell carcinoma, hairy cell leukemia, Familial Mediterranean Fever, andCastleman's Disease. Subjects that may benefit from the disclosedprophylactic methods include those subjects susceptible to or at risk ofdeveloping any of the foregoing disorders.

Also provided are methods of therapeutically treating orprophylactically treating a subject having amyloidosis associated withan aggregated amyloid protein comprising the amino acid sequence EDusing an antibody or antigen-binding fragment that specifically binds toan epitope comprising X₁EDX₂ in an aggregated amyloid protein, whereinX₁ and X₂ are any amino acid. Subjects that may benefit from thedisclosed therapeutic methods of treating amyloidosis associated with anaggregated amyloid protein include those subjects suffering from AAamyloidosis, AL amyloidosis, Alzheimer's disease, Mild CognitiveImpairment, amyloid polyneuropathy, Mediterranean fever, Muckle-Wellssyndrome, reactive systemic amyloidosis associated with systemicinflammatory diseases, myeloma or macroglobulinemia associatedamyloidosis, amyloidosis associated with immunocyte dyscrasia,monoclonal gammopathy, occult dyscrasia, and local nodular amyloidosisassociated with chronic inflammatory diseases. Subjects that may benefitfrom the disclosed prophylactic methods include those subjectssusceptible to or at risk of developing any of the foregoing disorders.In one aspect of the invention, the amyloid protein comprises thesequence AEDV (SEQ ID NO: 23), and the amyloidogenic disease treatedtherapeutically or prophylactically using the disclosed methods is AAamyloidosis, AL amyloidosis, amyloid polyneuropathy, Mediterraneanfever, Muckle-Wells syndrome, reactive systemic amyloidosis associatedwith systemic inflammatory diseases, myeloma or macroglobulinemiaassociated amyloidosis, amyloidosis associated with immunocytedyscrasia, monoclonal gammopathy, occult dyscrasia, and local nodularamyloidosis associated with chronic inflammatory diseases.

The disclosed therapeutic and prophylactic methods are useful fortreating human subjects.

Representative indices of efficacious therapeutic treatment includeslowing the progression of amyloidosis, inhibiting deposition of amyloidfibril aggregates, and/or clearing of amyloid fibril aggregates.Representative indices of efficacious prophylactic treatment includedelaying onset of amyloidosis and/or reducing a risk of amyloidosis.

Still further provided are methods of detecting an amyloid depositassociated with AA amyloidosis in a subject human, humanized, orchimeric antibody, or antigen-binding fragment thereof, thatspecifically binds to an epitope within residues 70-76 of human amyloidA peptide, which antibody or antigen-binding fragment is bound to adetectable label, and then detecting the detectable label in thesubject. Additional methods comprise detecting an aggregated amyloidprotein comprising the amino acid sequence ED using an antibody orantigen-binding fragment that specifically binds to an epitopecomprising X₁EDX₂ in an aggregated amyloid protein, wherein X₁ and X₂are any amino acid. The foregoing detection methods may be used, forexample, for monitoring onset or progression of disease or therapy inany of the above-noted diseases and disorders. As for the treatmentmethods disclosed herein, such monitoring may be performed in humans aswell as non-human subjects. Useful detectable labels includeradiolabels, such as ¹²⁵I. In performing such detection methods, thestep of detecting the detectable label may be accomplished bynon-invasive techniques, such as SPECT/CT imaging and NMR spectroscopy.

Still further provided are methods of active immunotherapy of a subjecthaving AA amyloidosis using an agent that induces an immune response toresidues 70-76 of amyloid A peptide effective to induce an immuneresponse comprising antibodies against residues 70-76 of an amyloid Apeptide. Representative agents for inducing the immune response includeresidues 70-76 of amyloid A peptide or a subfragment of at least 3contiguous residues thereof having fewer than 20 contiguous amino acidsfrom an AA peptide. These methods are useful both therapeutically and/orprophylactically for treatment of the subjects described herein abovewith respect to passive immunotherapy, i.e., by administering anantibody or antigen-binding fragment that specifically binds to residues70-76 of amyloid A peptide. Indices of therapeutic and prophylacticefficacy are also as noted herein above with respect to passiveimmunotherapy.

The foregoing summarizes particular aspects of the invention, andadditional aspects of the invention are described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Sequence alignment of human SAA1, human SAA2, human SAA3 andhuman SAA4.

FIG. 2: Sequence alignment of human SAA1 and human AA1.

FIG. 3: Sequence alignment of human SAA2 and human AA2.

FIG. 4: Sequence alignment t of human SAA3 and human AA3.

FIG. 5: Sequence alignment of human SAA4 and hHuman AA4.

FIG. 6: Sequence alignment of human AA1, human AA2, human AA3 and humanAA4.

FIG. 7: Sequence alignment of the last seven residues of human AA1,hHuman AA2, human AA3 and human AA4.

FIG. 8: Sequence alignment of mouse SAM, mouse SAA2, mouse SAA3 andmouse SAA4.

FIG. 9: Sequence alignment of mouse SAA1 and mouse AA1.

FIG. 10: Sequence alignment of mouse SAA2 and mouse AA2.

FIG. 11: Sequence alignment of mouse SAA3 and mouse AA3.

FIG. 12: Sequence alignment of mouse SAA4 and mMouse AA4.

FIG. 13: Sequence alignment of mouse AA1, mouse AA2, mouse AA3 mouseAA4.

FIG. 14: Sequence alignment of the last seven residues of mouse AA1,mouse AA2, mouse AA3 mouse AA4.

FIG. 15: Sequence alignment of human SAA1 and mouse SAA1.

FIG. 16: Sequence alignment of human AA1 and mouse AA1.

FIG. 17: Sequence alignment of human SAA1 and mouse SAA1 Fragment.

FIG. 18: Sequence alignment of human SAA1 alpha, human SAA1 beta, andhuman SAM gamma.

FIG. 19: Sequence alignment of human SAA2 alpha and human SAA2 beta.

FIG. 20: Sequence comparison of SAA proteins. The peptide region used togenerate 2A4, 8G9 and 7D8 is shown in dashed lines. The 8 amino acidinsert between positions 67 and 68 in the Shar Pei sequence is indicatedby the underline and arrow. Alignment performed with CLUSTALW.

FIG. 21: Germline sequences of Vκ light chains.

FIG. 22: Germline sequences of Vλ light chains.

FIG. 23: Amino acid sequence of Vλ6 Wil.

FIG. 24: X-ray crystal of Vλ6 Wil showing position of Glu50-Asp51.

FIG. 25: X-ray crystal of Vλ6 Wil showing position of Glu81-Asp82

FIG. 26: Binding kinetics of Elan mAbs to synthetic Vλ6 Wil fibrils.BIAcore measurements of the interaction of mAbs 2A4, 7D8 and 8G9 at 6.6nM to immobilized Vλ6 Wil fibrils. The calculated KD for eachinteraction was ˜1 nM.

FIG. 27: Concentration-dependent binding kinetics of mAb 7D8 tosynthetic Vλ6 Wil fibrils. The antibody interaction at a concentrationof 6.6-33.3 nM to immobilized Vλ6 Wil fibrils was measured by BIAcore.

FIG. 28: Binding kinetics of mAb 7D8 to synthetic V_(λ)6 Wil fibrils inthe presence of the p39 and p41 peptides. The interaction of the mAb 7D8at 6.6 nM with immobilized V_(λ)6 Will fibrils was measured by BIAcorein the presence of peptides p39 and p41 at 1 or 20 μg/mL.

FIG. 29: Reactivity of monoclonal antibodies with ALλ tissue amyloiddeposits.

FIG. 30: Biodistribution of ¹²⁵I-labeled mAb 7D8 in mice bearing a humanALλ amyloidoma.

FIG. 31: Interaction of anti-AA of culture supernatants withmurine-derived AA fibrils. Results of mAb culture supernatants bindingmurine AA AEF. Upper and lower panels are data on first and secondculture fluid harvest, respectively.

FIG. 32: SDS-PAGE analysis of protein A-purified 2A4, 8G9 and 7D8 mAbs.

FIG. 33: Binding of purified mAbs to immunizing (p#39) and controlpeptide (p#41).

FIG. 34: Binding to murine AA amyloid extract (AEF).

FIG. 35: Binding of purified mAbs to human renal AA amyloid extract.

FIGS. 36A-36E: Sequences of murine 2A4, 7D8, and 8G9 light chain andheavy chain variable regions (FIG. 36A); sequences of humanized 2A4/8G9and 7D8 light chain variable regions (FIGS. 36B-36C); sequences of humanlight chain variable regions used as acceptor frameworks (FIG. 36D);sequences of humanized 2A4/7D8/8G9 heavy chain variable regions andhuman heavy chain variable region used as acceptor framework (FIG. 36E).Underlining, CDRs; double underlining, leader sequences; lower case,back mutations.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides an isolated antibody or antigen-binding fragmentthereof, which specifically binds to an epitope including X₁EDX₂ in anaggregated amyloid protein, wherein X₁ and X₂ are any amino acid.

Representative antibodies of the invention also include antibodies orfragments thereof that (a) compete for binding to an epitope includingX₁EDX₂ with a 2A4, 7D8, or 8G9 antibody; (b) bind to the same epitopeincluding X₁EDX₂ as a 2A4, 7D8, or 8G9 antibody; (c) include an antigenbinding domain of a 2A4, 7D8, or 8G9 antibody; or (d) include the sixcomplementarity determining regions (CDRs) of a 2A4, 7D8, or 8G9antibody.

The invention also provides an isolated antibody variable regionincluding (a) a light chain variable region of an antibody derived froma 2A4, 7D8, or 8G9 antibody; or (b) a heavy chain variable region of anantibody derived from a 2A4, 7D8, or 8G9 antibody.

The invention also provides an isolated nucleic acid encoding anantibody light chain variable region or heavy chain variable regionincluding (a) a nucleotide sequence that encodes a light chain or heavychain variable region of a 7D8, 2A4, or 8G9 antibody; (b) a nucleotidesequence that is identical to a nucleotide sequence of a 7D8, a 2A4, oran 8G9 antibody that encodes a light chain or heavy chain variableregion; (c) a nucleotide sequence that is substantially identical to anucleotide sequence of (a) or (b); or (d) a nucleic acid thatspecifically hybridizes to a nucleic acid having a nucleotide sequencethat is the complement of a nucleotide sequence of (a) or (b) understringent hybridization conditions.

Cells expressing the antibodies and antigen-binding fragments of thepresent invention are also provided. The invention further providescells expressing nucleic acids of the invention.

The invention also includes methods of treating amyloid diseases andmethods of prophylaxis of amyloid diseases using the antibodies andantigen-binding fragments of the invention. Currently, there are noapproved specific amyloid-directed treatments for any of the amyloiddiseases, including AA Amyloidosis and AL amyloidosis. See Gillmore J.D. et al., Lancet 358:24-9 (2001). Where there is an underlying orassociated disease state, therapy directed towards decreasing theproduction of amyloidogenic protein by treating the underlying disease.For example, current treatment strategy for AA Amyloidosis is to targetunderlying inflammation, reducing ApoSSA levels to below 10 mg/l.Currently employed therapies include chemotherapy (cholorambucil andMTX), immuno-suppressants (azathioprine), anti-inflammatory drugs(colchicine) and TNF inhibitors. The invention provides pharmaceuticalcompositions and methods for treating a number of amyloid diseases,including amyloidosis, such as, for example, AA amyloidosis and ALamyloidosis. According to one aspect, the invention includespharmaceutical compositions that include, as an active ingredient, anagent that is effective to induce an immune response in a patientagainst an amyloid component. The agent can be a peptide comprising afragment consisting of the amino acid sequence X₁EDX₂ derived from anamyloid protein. The agent can be an antibody that specifically binds toan epitope comprising X₁EDX₂. In other embodiments, the agent can be anantigen-binding fragment of an antibody. Such compositions willgenerally also include excipients and in preferred embodiments mayinclude adjuvants. In further preferred embodiments, the adjuvantsinclude, for example, aluminum hydroxide, aluminum phosphate, MPL™,QS-21 (STIMULON™) or incomplete Freund's adjuvant. According to arelated embodiment, such pharmaceutical compositions may include aplurality of agents effective to induce an immune response against morethan one amyloid component in the patient.

In a related embodiment, the agent is effective to produce an immuneresponse directed against an aggregated amyloid protein, such as afibril peptide or protein amyloid component. Preferably, such a fibrilpeptide or protein is derived from a fibril precursor protein known tobe associated with certain forms of amyloid diseases, as describedherein. Such precursor proteins include, but are not limited to, SerumAmyloid A protein (ApoSSA), immunoglobulin light chain, immunoglobulinheavy chain, ApoAI, transthyretin, lysozyme, fibrogen α chain, gelsolin,cystatin C, Amyloid β protein precursor (β-APP), Beta₂ microglobulin,prion precursor protein (PrP), atrial natriuretic factor, keratin, isletamyloid polypeptide, a peptide hormone, and synuclein. Such precursorsalso include mutant proteins, protein fragments and proteolytic peptidesof such precursors. In a preferred embodiment, the agent is effective toinduce an immune response directed against a neoepitope formed by afibril protein or peptide, with respect to a fibril precursor protein.That is, as described in more detail herein, many fibril-formingpeptides or proteins are fragments of such precursor proteins, such asthose listed above. When such fragments are formed, such as byproteolytic cleavage, epitopes may be revealed that are not present onthe precursor and are therefore not immunologically available to theimmune system when the fragment is a part of the precursor protein.Agents directed to such epitopes may be preferred therapeutic agents,since they may be less likely to induce an autoimmune response in thepatient. Preferably, such agents preferentially produce an immuneresponse directed against a pathological form of the amyloid protein,for example, an aggregated amyloid protein, relative to nonpathologicalforms of the amyloid protein.

According to a related embodiment, pharmaceutical compositions of theinvention include agents directed to amyloid aggregates, such as thoseselected from the group including, but not limited to the followingaggregated (e.g., fibril) peptides or proteins: AA, AL, ATTR, AApoA1,Alys, Agel, Acys, Aβ, AB₂M, AScr, Acal, AIAPP and synuclein-NACfragment. The full names and compositions of these peptides aredescribed herein. Such peptides can be made according to methods wellknown in the art, as described herein.

The methods comprise administering to the patient an effective dosage ofan antibody that specifically binds to an epitope comprising X₁EDX₂ inan amyloid protein, wherein X₁ is H, T, F, S, P, A or any other aminoacid residue immediately preceding ED in such amyloid protein; andwherein X₂ is T, S, E, R, I, V, F, A or any other amino acid residueimmediately following ED in such amyloid protein. In some methods, thepatient is suffering from an amyloidosis associated with an aggregatedamyloid protein comprising the amino acid sequence ED. Some antibodiesspecifically bind to an epitope consisting of such X₁EDX₂. In someantibodies, X₁ is H, T, F, S, P, or A and X₂ is T, S, E, D, R, I, V, For A. In some such antibodies, when X₁ is H, X₂ is T or A; when X₁ is A,X₂ is S, T, E or V; when X₁ is T, X₂ is E; when X₁ is F, X₂ is D; whenX₁ is S, X₂ is E, F or A; and when X₁ is P, X₂ is E, I or F. In someantibodies, X₁ is H, T, F, S, P, or A and X₂ is T, S, E, D, R, I, V, For A, with the proviso that if X₁ is A, X₂ is not V. In some antibodies,when X₁ is A, X₂ is S, T or E.

Some antibodies specifically bind an epitope comprising the amino acidsequence GHEDT, (SEQ ID NO: 3), HEDT, (SEQ ID NO: 12), AEDS, (SEQ ID NO13), AEDT (SEQ ID NO: 14), HEDA (SEQ ID NO: 15), TEDE, (SEQ ID NO: 16),FEDD, (SEQ ID NO: 17), SEDE, (SEQ ID NO: 18), AEDE, (SEQ ID NO: 19),PEDE, (SEQ ID NO: 20), PEDI, (SEQ ID NO: 21), PEDF, (SEQ ID NO: 22),AEDV, (SEQ ID NO: 23), SEDF (SEQ ID NO: 24), or SEDA, (SEQ ID NO: 25).

Some antibodies specifically bind to a peptide comprising an amino acidsequence selected from the group consisting of GHEDT, (SEQ ID NO: 3),HEDT, (SEQ ID NO: 12), AEDS, (SEQ ID NO: 13), AEDT, (SEQ ID NO: 14),HEDA, (SEQ ID NO: 15), TEDE, (SEQ ID NO: 16), FEDD, (SEQ ID NO: 17),SEDE, (SEQ ID NO: 18), AEDE, (SEQ ID NO: 19), PEDE, (SEQ ID NO: 20),PEDI, (SEQ ID NO: 21), PEDF, (SEQ ID NO: 22), SEDF, (SEQ ID NO: 24), andSEDA, (SEQ ID NO: 25). Some antibodies specifically bind to a peptidecomprising an amino acid sequence selected from the group consisting ofGHEDT, (SEQ ID NO: 3, HEDT, (SEQ ID NO: 12), AEDS, (SEQ ID NO: 13),AEDT, (SEQ ID NO: 14), HEDA, (SEQ ID NO: 15), and TEDE, (SEQ ID NO: 16).

Some antibodies specifically bind to an epitope within residues 70 to 76of AA. Some antibodies specifically bind to an epitope within residues71 to 75 of AA. Some antibodies are raised to a peptide comprisingGHEDT, (SEQ ID NO: 3).

Some antibodies specifically bind to a peptide comprising the amino acidsequence PEDS, (SEQ ID NO: 26), PEDL, (SEQ ID NO: 27), TEDV, (SEQ ID NO:28), AEDE, (SEQ ID NO: 19), SEDI, (SEQ ID NO: 29), and TEDT, (SEQ ID NO:30). Some antibodies specifically bind to a peptide comprising the aminoacid sequence LEDG, (SEQ ID NO: 31), AEDM, (SEQ ID NO: 32), HEDS, (SEQID NO: 33), CEDD, (SEQ ID NO: 34), QEDS, (SEQ ID NO: 35), REDS, (SEQ IDNO: 36), TEDG, (SEQ ID NO: 37), QEDR, (SEQ ID NO: 38), TEDL, (SEQ ID NO:39), PEDN, (SEQ ID NO: 40), EEDP, (SEQ ID NO: 41), LEDL, (SEQ ID NO:42), KEDA, (SEQ ID NO: 43), SEDC, (SEQ ID NO: 44), EEDD, (SEQ ID NO:45), SEDK, (SEQ ID NO: 46), DEDD, (SEQ ID NO: 47), DEDG, (SEQ ID NO:48), LEDE, (SEQ ID NO: 49), GEDA, (SEQ ID NO: 50), VEDF, (SEQ ID NO:51), YEDE, (SEQ ID NO: 52), IEDL, (SEQ ID NO: 53), WEDY, (SEQ ID NO:54), DEDW, (SEQ ID NO: 55), SEDL, (SEQ ID NO: 56), YEDQ, (SEQ ID NO:57), LEDW, (SEQ ID NO: 58), YEDR, (SEQ ID NO: 59), and PEDK, (SEQ ID NO:60).

Some antibodies specifically bind to a peptide comprising the amino acidsequence AEDV, (SEQ ID NO: 23). Some antibodies specifically bind to apeptide comprising the amino acid sequence SEDF, (SEQ ID NO: 24) orPEDF, (SEQ ID NO: 22). Some antibodies specifically bind to a peptidecomprising the amino acid sequence AEDS, (SEQ ID NO: 13). Someantibodies specifically bind to a peptide comprising the amino acidsequence PEDI (SEQ ID NO: 21), AEDV, (SEQ ID NO 23), SEDF, (SEQ ID NO:24), SEDA, (SEQ ID NO: 25), SEDE, (SEQ ID NO: 18), AEDE, (SEQ ID NO:19), and PEDE, (SEQ ID NO: 20). Some antibodies bind to a peptidecomprising the amino acid sequence TEDE, (SEQ ID NO: 16).

Some antibodies specifically bind to a peptide comprising the amino acidsequence AEDV, (SEQ ID NO: 23). Some antibodies specifically bind to apeptide comprising the amino acid sequence SEDF, (SEQ ID NO: 24) orPEDF, (SEQ ID NO: 22). Some antibodies specifically bind to a peptidecomprising the amino acid sequence AEDS, (SEQ ID NO: 13). Someantibodies specifically bind to a peptide comprising the amino acidsequence PEDI (SEQ ID NO: 21), AEDV, (SEQ ID NO 23), SEDF, (SEQ ID NO:24), SEDA, (SEQ ID NO: 25), SEDE, (SEQ ID NO: 18), AEDE, (SEQ ID NO:19), and PEDE, (SEQ ID NO: 20). Some antibodies bind to a peptidecomprising the amino acid sequence TEDE, (SEQ ID NO: 16).

Any of the antibodies described above can be administered in the methodsdescribed above to treat or effect prophylaxis of a diseasecharacterized by the deposition of an amyloid protein, such as, forexample, an amyloid protein comprising the amino acid sequence ED. Insome methods, if the amyloid protein comprises the amino acid sequenceAEDV, (SEQ ID NO: 23), then the antibody is not administered to treat oreffect prophylaxis of Alzheimer's disease or Mild Cognitive Impairment.The amyloid protein can be any of serum amyloid A protein,immunoglobulin light chain protein, such as, for example, Vλ6 Wil or Vκ,human islet amyloid precursor polypeptide (IAPP), beta amyloid peptide,transthyretin (TTR) or ApoA1.

Optionally, the patient is human. Optionally, the antibody specificallybinds to a peptide whose residues consist of SEQ ID NOS. 4, 5, 6, 7, 8,9, 10, or 11. Optionally, the antibody specifically binds to an epitopewithin residues 70-76 of (SEQ ID NO: 2). Optionally, the antibody is ahuman antibody, humanized antibody or chimeric antibody. Optionally, thehuman antibody is of human isotype IgG1, IgG4, IgG2 or IgG3. Optionally,the humanized antibody is of human isotype IgG1, IgG4, IgG2 or IgG3.Optionally, the chimeric antibody is of human isotype IgG1, IgG4, IgG2or IgG3. Optionally, the antibody is a mouse antibody. Optionally, theantibody is a polyclonal antibody. Optionally, the antibody is amonoclonal antibody.

In some treatment methods, the antibody comprises two copies of the samepair of light and heavy chains. In other methods, the antibody is abispecific antibody comprising a first light and heavy chain pair thatspecifically binds to the epitope of Aβ and a second light and heavychain pair that specifically binds to an Fc receptor on microglialcells. In other methods, a chain of the antibody is fused to aheterologous polypeptide.

Some treatment methods, the dosage of antibody is at least 1 mg/kg bodyweight of the patient. In other methods, the dosage of antibody is atleast 10 mg/kg body weight of the patient.

In some treatment methods, the antibody is administered with a carrieras a pharmaceutical composition. In other methods, wherein the antibodyis a human antibody to AA prepared from B cells from a human immunizedwith an AA peptide. Optionally, the human immunized with AA peptide isthe patient. In some methods, the antibody is administeredintraperitoneally, orally, intranasally, subcutaneously,intramuscularly, topically or intravenously.

In some treatment methods, the antibody is administered by administeringa polynucleotide encoding at least one antibody chain to the patient andthe polynucleotide is expressed to produce the antibody chain in thepatient. Optionally, the polynucleotide encodes heavy and light chainsof the antibody and the polynucleotide is expressed to produce the heavyand light chains in the patient.

Some of the above treatment methods further comprise administering aneffective dosage of at least one other antibody that binds to adifferent epitope of AA. Some of the above treatment methods furthercomprise monitoring the patient for level of administered antibody inthe blood of the patient. In other methods, the antibody is administeredin multiple dosages over a period of at least six months. In othermethods, the antibody is administered as a sustained releasecomposition.

The invention further provides methods of effecting prophylaxis of AAamyloidosis in a patient susceptible to AA amyloidosis. The methodscomprise administering to the patient an effective dosage of an antibodythat specifically binds to an epitope within residues 70 to 76 of AA.Optionally, the patient is human. Optionally, the antibody specificallybinds to a peptide whose residues consist of SEQ ID NOS. 4, 5, 6, 7, 8,9, 10, or 11. Optionally, the antibody specifically binds to an epitopewithin residues 70-76 of (SEQ ID NO: 2). In some methods, the patientsuffers from an underlying amyloid disease selected from the groupconsisting of rheumatoid arthritis, juvenile chronic arthritis,ankylosing spondylitis, psoriasis, psoriatic arthropathy, Reiter'ssyndrome, Adult Still's disease, Behcet's syndrome, Crohn's disease,leprosy, tuberculosis, bronchiectasis, decubitus ulcers, chronicpyelonephritis, osteomyelitis, Whipple's disease, Hodgkin's lymphoma,renal carcinoma, carcinomas of gut, lung and urogenital tract, basalcell carcinoma, hairy cell leukemia, Familial Mediterranean Fever, andCastleman's Disease.

The invention further provides a human, humanized, or chimeric antibodythat specifically binds to an epitope within residues 70 to 76 of AA.Optionally, the humanized antibody specifically binds to an epitopewithin residues 70 to 76 of AA. Optionally, the humanized antibody is ahumanized version 7D8 antibody (ATCC Accession Number PTA-9468).Optionally, the humanized antibody is a humanized version 7D29 antibody.Optionally, the humanized antibody is a humanized version 7D19 antibody.Optionally, the humanized antibody is a humanized version 7D47 antibody.Optionally, the humanized antibody is a humanized version 7D39 antibody.Optionally, the humanized antibody is a humanized version 7D66 antibody.Optionally, the humanized antibody is a humanized version 8G9 antibody.Optionally, the humanized antibody is a humanized version 8G3 antibody.Optionally, the humanized antibody is a humanized version 8G4 antibody.Optionally, the humanized antibody is a humanized version 8G51 antibody.Optionally, the humanized antibody is a humanized version 8G22 antibody.Optionally, the humanized antibody is a humanized version 8G30 antibody.Optionally, the humanized antibody is a humanized version 8G46 antibody.Optionally, the humanized antibody is a humanized version 2A4 antibody(ATCC Accession Number PTA-9662). Optionally, the humanized antibody isa humanized version 2A20 antibody. Optionally, the humanized antibody isa humanized version 2A44 antibody. Optionally, the humanized antibody isa humanized version 2A77 antibody. Optionally, the humanized antibody isa humanized version 2A13 antibody. Optionally, the humanized antibody isa humanized version 2A14 antibody.

The invention further provides pharmaceutical compositions. Thepharmaceutical compositions comprise an antibody that specifically bindsto an epitope within residues 70 to 76 of AA, and a pharmaceuticallyacceptable carrier. Some pharmaceutical compositions comprise a human,humanized, or chimeric antibody that specifically binds to an epitopewithin residues 70 to 76 of AA, and a pharmaceutically acceptablecarrier. Other pharmaceutical compositions comprise an antibody thatspecifically binds to an epitope within residues 70 to 76 of AA and apharmaceutically acceptable carrier, where the isotype of the antibodyis human IgG1, and a pharmaceutically acceptable carrier. In somepharmaceutical compositions the isotype of the antibody is human IgG2,IgG3, or IgG4. In some pharmaceutical compositions the antibody ishuman. In some pharmaceutical compositions the antibody is humanized. Insome pharmaceutical compositions the antibody is chimeric. In somepharmaceutical compositions the antibody is a polyclonal antibody. Insome pharmaceutical compositions the antibody is a monoclonal antibody.

In some pharmaceutical compositions the antibody comprises two copies ofthe same pair of light and heavy chains. In some pharmaceuticalcompositions the antibody is a bispecific antibody comprising a firstlight and heavy chain pair that specifically binds to the epitope of AAand a second light and heavy chain pair that specifically binds to an Fcreceptor on microglial cells. In some pharmaceutical compositions achain of the antibody is fused to a heterologous polypeptide. In somepharmaceutical compositions the carrier is a physiologically acceptablediluent for parenteral administration. Some pharmaceutical compositionsare adapted to be administered intraperitoneally, orally, intranasally,subcutaneously, intramuscularly, topically or intravenously. Somepharmaceutical compositions are adapted to be administered in multipledosages over a period of at least six months. Some pharmaceuticalcompositions are adapted to be administered as a sustained releasecomposition. Some pharmaceutical compositions further comprise at leastone other antibody that binds to a different epitope of AA.

The invention provides methods of treating AA amyloidosis in a patient.The methods comprise administering an agent that induces an immuneresponse to AA70-76 in a regime effective to induce an immune responsecomprising antibodies against AA70-76 in a regime effective to induce animmune response comprising antibodies against AA70-76. In some methodsthe patient is human. Optionally, the agent comprises AA70-76 or asubfragment of at least 3 contiguous residues thereof and has fewer than20 contiguous amino acids from an AA peptide. Optionally, the agent is apeptide having a sequence selected from the group consisting of SEQ IDNOS 4, 5, 6, 7, 8, 9, 10 and 11. and subfragments of at least 3contiguous residues thereof and has fewer than 20 amino acids from an AApeptide. Optionally, the agent is linked at its N and C termini to firstand second heterologous polypeptides. Optionally, the agent is linked atits N terminus to a heterologous polypeptide, and at its C-terminus toat least one additional copy of the N-terminal segment. In some methodsthe heterologous polypeptide induces a T-cell response against theheterologous polypeptide and thereby a B-cell response against AA. Insome methods the polypeptide further comprises at least one additionalcopy of AA. Optionally, the polypeptide comprises from N-terminus toC-terminus, AA, a plurality of additional copies of AA, and theheterologous amino acid segment.

In some treatment methods the polypeptide is administered with anadjuvant that enhances an immune response to the N-terminal segment.Optionally, the adjuvant and the polypeptide are administered togetheras a composition. Optionally, the adjuvant is administered before thepolypeptide. Optionally, the adjuvant is administered after thepolypeptide. In some methods the adjuvant is alum. In some methods theadjuvant is MPL. In some methods the adjuvant is QS-21. In some methodsthe adjuvant is incomplete Freund's adjuvant. In some methods the immuneresponse comprises T-cells that bind to the AA peptide as a component ofan MHC I or MHC II complex.

The invention provides methods of effecting prophylaxis of AAamyloidosis in a patient. The methods comprise administering an agentthat induces an immune response to AA70-76 in a regime effective toinduce an immune response comprising antibodies against AA70-76 in aregime effective to induce an immune response comprising antibodiesagainst AA70-76. In some methods the patient is human. In some methodsthe patient is asymptomatic. In some methods the patient suffers from anunderlying amyloid disease selected from the group consisting ofrheumatoid arthritis, juvenile chronic arthritis, ankylosingspondylitis, psoriasis, psoriatic arthropathy, Reiter's syndrome, AdultStill's disease, Behcet's syndrome, Crohn's disease, leprosy,tuberculosis, bronchiectasis, decubitus ulcers, chronic pyelonephritis,osteomyelitis, Whipple's disease, Hodgkin's lymphoma, renal carcinoma,carcinomas of gut, lung and urogenital tract, basal cell carcinoma,hairy cell leukemia, Familial Mediterranean Fever, and Castleman'sDisease.

In some methods of effecting prohylaxis, the agent comprises AA70-76 ora subfragment of at least 3 contiguous residues thereof and has fewerthan 20 contiguous amino acids from an AA peptide. Optionally, the agentis a peptide having a sequence selected from the group consisting of SEQID NOS 4, 5, 6, 7, 8, 9, 10 and 11. and subfragments of at least 3contiguous residues thereof and has fewer than 20 amino acids from an AApeptide. Optionally, the agent is linked at its N and C termini to firstand second heterologous polypeptides. Optionally, the agent is linked atits N terminus to a heterologous polypeptide, and at its C-terminus toat least one additional copy of the N-terminal segment. In some methodsthe heterologous polypeptide induces a T-cell response against theheterologous polypeptide and thereby a B-cell response against AA. Insome methods the polypeptide further comprises at least one additionalcopy of AA. Optionally, the polypeptide comprises from N-terminus toC-terminus, AA, a plurality of additional copies of AA, and theheterologous amino acid segment.

The invention further provides pharmaceutical compositions. Thepharmaceutical compositions comprise an AA fragment consisting ofresidues beginning at residue 70 of AA and ending at residue 76 of AA.Optionally, the AA fragment is linked at its C-terminus to aheterologous polypeptide. Optionally, the AA fragment is linked at itsN-terminus to a heterologous polypeptide. Optionally, the AA fragment islinked at its N and C termini to first and second heterologouspolypeptides. Optionally, the AA fragment is linked at its N terminus toa heterologous polypeptide, and at its C-terminus to at least oneadditional copy of the N-terminal segment. Optionally, the polypeptidefurther comprises at least one additional copy of the N-terminalsegment. Optionally, the polypeptide comprises from N-terminus toC-terminus, AA, a plurality of additional copies of the N-terminalsegment, and the heterologous amino acid segment. In some pharmaceuticalcompositions the heterologous polypeptide induces a T-cell responseagainst the heterologous polypeptide and thereby a B-cell responseagainst the N-terminal segment.

Some pharmaceutical compositions further comprise an adjuvant thatenhances an immune response to AA. Optionally, the adjuvant is alum.Optionally, the adjuvant is MPL. Optionally, the adjuvant is QS-21.Optionally, the adjuvant is incomplete Freund's adjuvant. Optionally,the adjuvant further comprises GM-CSF. Optionally, the adjuvant isM-CSF. Optionally, the composition comprises greater than 10 microgramsof the polypeptide.

The invention provides methods of treating AA amyloidosis in a patient.The methods comprise administering an agent effective to induce animmune response against a peptide component of an amyloid deposit in thepatient and a different agent that treats an underlying disease, andthereby treating AA amyloidosis in the patient. In some methods theunderlying disease is selected from the group consisting of rheumatoidarthritis, juvenile chronic arthritis, ankylosing spondylitis,psoriasis, psoriatic arthropathy, Reiter's syndrome, Adult Still'sdisease, Behcet's syndrome, Crohn's disease, leprosy, tuberculosis,bronchiectasis, decubitus ulcers, chronic pyelonephritis, osteomyelitis,Whipple's disease, Hodgkin's lymphoma, renal carcinoma, carcinomas ofgut, lung and urogenital tract, basal cell carcinoma, hairy cellleukemia, Familial Mediterranean Fever, and Castleman's Disease.

The invention provides methods of effecting prophylaxis of AAamyloidosis in a patient. The methods comprise administering an agenteffective to induce an immune response against a peptide component of anamyloid deposit in the patient and a different agent that treats anunderlying disease, and thereby treating AA amyloidosis in the patient.In some methods the underlying disease is selected from the groupconsisting of rheumatoid arthritis, juvenile chronic arthritis,ankylosing spondylitis, psoriasis, psoriatic arthropathy, Reiter'ssyndrome, Adult Still's disease, Behcet's syndrome, Crohn's disease,leprosy, tuberculosis, bronchiectasis, decubitus ulcers, chronicpyelonephritis, osteomyelitis, Whipple's disease, Hodgkin's lymphoma,renal carcinoma, carcinomas of gut, lung and urogenital tract, basalcell carcinoma, hairy cell leukemia, Familial Mediterranean Fever, andCastleman's Disease.

The invention provides methods of screening an antibody for activity intreating a patient having AA amyloidosis. The methods comprisecontacting the antibody with AA peptide and determining whether theantibody specifically binds to AA, specific binding providing anindication that the antibody has activity in treating AA amyloidosis.

The invention provides methods of screening an antibody for activity inclearing a biological entity physically associated with an antigen. Themethods comprise combining the antigen-associated biological entity, theantibody and phagocytic cells bearing Fc receptors in a medium; andmonitoring the amount of the antigen-associated biological entityremaining in the medium, a reduction in amount of the antigen-associatedbiological entity indicating the antibody has clearing activity againstthe antigen. In some methods the monitoring step monitors the amount ofthe antigen remaining in the medium. In some methods the combiningcomprises adding antigen-associated biological entity to the medium, andcontacting the medium with the phagocytic cells bearing Fc receptors. Insome methods the antigen-associated biological entity is provided as atissue sample. In some methods the antigen is the biological entity. Insome methods the tissue sample comprises an amyloid deposit. Optionally,the tissue sample is from the patient or a mammal having AA Amyloidosispathology. In some methods, the antigen is AA. In some methods thephagocytic cells are microglial cells. In some methods the tissue sampleis selected from the group consisting of a cancerous tissue sample, avirally infected tissue sample, a tissue sample comprising inflammatorycells, a nonmalignant abnormal cell growth, and a tissue samplecomprising an abnormal extracellular matrix.

The invention provides methods of detecting an amyloid deposit in apatient. The methods comprise administering to the patient an antibodythat specifically binds to an epitope within amino acids 70-76 of AA anddetecting presence of the antibody in the patient. Optionally, theantibody is labeled. Optionally, the antibody is labeled with aparamagnetic label. Optionally, the labeled antibody is detected bynuclear magnetic resonance. Optionally, the labeled antibody is detectedwith SPECT/CT imaging. In some methods, the antibody lacks capacity toinduce a clearance response on binding to an amyloid deposit in thepatient.

The invention provides diagnostic kits. The kits comprise an antibodythat specifically binds to an epitope with residues 70-76 of AA. Somekits further comprise labeling describing use of the antibody for invivo diagnosis or monitoring of a disease associated with amyloiddeposits of AA in a patient. In some embodiments, the kits includeinstructions for use of the antibody or antigen-binding fragment thereofin detecting AA.

The invention further provides a method of diagnosing amyloidosis in asubject comprising: (a) administering to the subject an antibody orantigen-binding fragment thereof that is bound to a detectable label,wherein the antibody or fragment thereof specifically binds to anepitope comprising X₁EDX₂ in an aggregated amyloid protein, wherein X₁and X₂ are any amino acid; and (b) detecting the presence or absence ofthe bound antibody or fragment thereof, wherein the presence of thebound antibody or fragment indicates a diagnosis of AA amyloidosis.

Further provided herein is a method of treatment or prophylaxis ofamyloidosis using an antibody or antigen-binding fragment thereof, whichspecifically binds to an epitope comprising X₁EDX₂ in an aggregatedamyloid protein, wherein X₁ and X₂ are any amino acid.

The present invention provides an antibody or antigen-binding fragmentthereof that binds specifically to an epitope comprising X₁EDX₂, in anaggregated amyloid protein, wherein X₁ and X₂ are any amino acid. Forexample, X₁ includes H, T, F, S, P, A, L, C, Q, R, E, K, D, G, V, Y, Ior W, such as H, T, F, S, P, or A, or such as H, T, F, or A. X₂ includesT, S, E, R, I, V, F, D, A, G, M, L, N, P, C, K, Y, or Q, such as T, S,E, R, I, V, F, D, or A, or such as T, S, E, D, or A. In other examples,X₁ is H, T, or A and X₂ is T, S, E, or A, such as X₁ is H or A and X₂ isT, S, or A. In yet additional examples, X₁ is H and H₂ is T or A; or X₁is A and X₂ is S, T, E, or V, such as X₁ is A and X₂ is S, T, or E, orX₁ is T and X₂ is E, or X₁ is F and X₂ is D, or X₁ is S and X₂ is E, F,or A; or X₁ is P and X₂ is E, I, or F.

In particular, the epitopes include amino acid sequences such as thoseset forth in SEQ ID NO: 3 through to SEQ ID NO: 25, such as SEQ ID NOS:3, 12, 13, 14, 15, and 16. Additional examples include SEQ ID NOS: 4, 5,7, 8, and 9, such as SEQ ID NO: 4. Antibodies of the invention that bindto the epitopes, such as to SEQ ID NO: 3, include the 2A4, 7D8, and 8G9antibodies.

The aggregated amyloid proteins to which antibodies of the inventionbind are non-monomeric proteins. Such aggregated amyloid proteinsinclude serum amyloid A protein (SAA), immunoglobulin light chainprotein, human islet amyloid precursor polypeptide (IAPP), beta amyloidpeptide, transthyretin (TTR), and ApoA1, such as SAA.

The invention further provides antibodies or antigen-binding fragmentsthereof that (a) compete for binding to an epitope that includes X₁EDX₂with a 2A4, 7D8, or 8G9 antibody; (b) bind to the same epitope thatincludes X₁EDX₂ as a 2A4, 7D8, or 8G9 antibody; (c) have anantigen-binding domain of a 2A4, 7D8, or 8G9 antibody; or (d) includethe six complementarity determining regions (CDRs) of a 2A4, 7D8, or 8G9antibody. The invention also provides chimeric or humanized versions ofa 2A4, 7D8, or 8G9 antibody.

Representative antibodies, which specifically bind to an epitope thatincludes X₁EDX₂, also include antibodies having at least one, two, orthree of the complementarity determining regions (CDRs) of a light chainof a 2A4, 7D8 or 8G9 antibody. Antibodies of the invention, whichspecifically bind to an epitope that includes X₁EDX₂, also includeantibodies having at least one, two, or three of the CDRs of a heavychain of a 2A4, 7D8, or 8G9 antibody.

CDRs can be identified according to methods known in the art. Forexample, numbering systems for identifying CDRs are in common use. TheKabat definition is based on sequence variability, and the Chothiadefinition is based on the location of the structural loop regions. TheAbM definition is a compromise between the Kabat and Chothia approaches.The CDRs of the light chain variable region are bounded by the residuesat positions 24 and 34 (CDR1-L), 50 and 56 (CDR2-L), and 89 and 97(CDR3-L) according to the Kabat, Chothia, or AbM algorithm. According tothe Kabat definition, the CDRs of the heavy chain variable region arebounded by the residues at positions 31 and 35B (CDR1-H), 50 and 65(CDR2-H), and 95 and 102 (CDR3-H) (numbering according to Kabat).According to the Chothia definition, the CDRs of the heavy chainvariable region are bounded by the residues at positions 26 and 32(CDR1-H), 52 and 56 (CDR2-H), and 95 and 102 (CDR3-H) (numberingaccording to Chothia). According to the AbM definition, the CDRs of theheavy chain variable region are bounded by the residues at positions 26and 35B (CDR1-H), 50 and 58 (CDR2-H), and 95 and 102 (CDR3-H) (numberingaccording to Kabat). See Martin et al. (1989) Proc. Natl. Acad. Sci. USA86: 9268-9272; Martin et al. (1991) Methods Enzymol. 203: 121-153;Pedersen et al. (1992) Immunomethods 1: 126; and Rees et al. (1996) InSternberg M. J. E. (ed.), Protein Structure Prediction, OxfordUniversity Press, Oxford, pp. 141-172.

The antibodies of the invention further include an antibody that bindsspecifically to an epitope comprising X₁EDX₂, in an aggregated amyloidprotein, wherein X₁ and X₂ are any amino acid, having variable regionsderived from variable regions of a 2A4, 7D8, or 8G9 antibody. Antibodieshaving variable regions of 2A4, 7D8, or 8G9 antibodies are alsoincluded.

The antibodies of the invention further include chimeric antibodies,human antibodies, humanized antibodies, single chain antibodies,tetrameric antibodies, tetravalent antibodies, multispecific antibodiesdomain-specific antibodies, domain-deleted antibodies or fusionproteins.

Fragments of the antibodies of the invention are also provided. Thefragments of the invention may be Fab fragments, Fab′ fragment, F(ab′)₂fragments, Fv fragments or ScFv fragments. Such antibodies or fragmentsthereof can be coupled with a cytotoxic agent, a radiotherapeutic agent,or a detectable label.

The invention also provides an isolated antibody variable regioncomprising (a) a light chain variable region derived from a 7D8, 2A4, or8G9 antibody light chain variable region, or (b) a heavy chain variableregion derived from a 7D8, 2A4, or 8G9 antibody light chain variableregion. Isolated variable regions are also provided having a light chainor heavy chain variable region of a 7D8, 2A4, or 8G9 antibody. Theisolated antibody variable regions are useful in antibody production.

The invention also provides isolated nucleic acids encoding an antibodylight chain variable region or a heavy chain variable region having (a)a nucleotide sequence that encodes a light chain or heavy chain variableregion of a 7D8, 2A4, or 8G9 antibody; (b) a nucleotide sequence that isidentical to a nucleotide sequence of a 7D8, a 2A4, or an 8G9 antibodythat encodes a light or heavy chain variable region; or (c) a nucleotidesequence that is substantially identical, i.e., at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.5%, or 99% to a nucleotide sequence of (a) or(b); or (d) a nucleic acid that specifically hybridizes to a nucleicacid having a nucleotide sequence that is the complement of a nucleotidesequence of (a) or (b) under stringent hybridization conditions, forexample, final wash conditions of 0.1×SSC at 65° C.

The present invention further provides cells and cell lines expressingthe antibodies or nucleic acids of the invention. Representative hostcells include mammalian and human cells, such as CHO cells, HEK-293cells, HeLa cells, CV-1 cells, and COS cells. Methods for generating astable cell line following transformation of a heterologous constructinto a host cell are known in the art. Representative non-mammalian hostcells include insect cells (Potter et al. (1993) Int. Rev. Immunol.10(2-3):103-112). Antibodies may also be produced in transgenic animals(Houdebine (2002) Curr. Opin. Biotechnol. 13(6):625-629) and transgenicplants (Schillberg et al. (2003) Cell Mol. Life. Sci. 60(3):433-45).

The invention also provides methods of treating or effecting prophylaxisof amyloidosis associated using immunogenic fragments of an amyloidprotein comprising X₁EDX₂, wherein X₁ is H, T, F, S, P, A or any otheramino acid residue immediately preceding ED in such amyloid protein; andwherein X₂ is T, S, E, R, I, V, F, A or any other amino acid residueimmediately following ED in such amyloid protein. Without wishing to bebound by a particular theory, it is believed that an epitope comprisingX₁EDX₂ can become exposed when an amyloid protein aggregates, orundergoes fibrillogenesis or otherwise enters a fibrillar structure,whether by cleavage from a larger precursor protein or by conformationalchange. For example, representative methods of treatment or prophylaxisof AA amyloidosis include administration of AA 70-76 fragments orimmunogenic fragments thereof. The invention also provides methods oftreating or effecting prophylaxis of amyloidosis associated withdeposition of amyloid protein using antibodies reactive with X₁EDX₂ inan aggregated amyloid protein, wherein X₁ is H, T, F, S, P, A or anyother amino acid residue immediately preceding ED in such aggregatedamyloid protein; and wherein X₂ is T, S, E, R, I, V, F, A or any otheramino acid residue immediately following ED in such aggregated amyloidprotein. Preferably, such antibodies are preferentially reactive withaggregated amyloid protein relative to non-pathological amyloid protein.For example, methods of treatment or prophylaxis of AA amyloidosisassociated with AA fibrils may include administration of antibodiesspecific for C-terminal region of AA fibrils (˜residues 70-76 of AA).The antibodies can inhibit formation of AA aggregates (e.g., fibrils) orresult in their disaggregation and clearance, thus treating or effectingprophylaxis of AA amyloidosis.

I. Definitions

The term “substantial identity” means that two peptide sequences, whenoptimally aligned, such as by the programs GAP or BESTFIT using defaultgap weights, share at least 65 percent sequence identity, preferably atleast 80 or 90 percent sequence identity, more preferably at least 95percent sequence identity or more (e.g., 99 percent sequence identity orhigher).

Preferably, residue positions, which are not identical differ byconservative amino acid substitutions.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., supra). One example of algorithm that is suitable fordetermining percent sequence identity and sequence similarity is theBLAST algorithm, which is described in Altschul et al., J. Mol. Biol.215:403-410 (1990). Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). Typically, default program parameterscan be used to perform the sequence comparison, although customizedparameters can also be used. For amino acid sequences, the BLASTPprogram uses as defaults a wordlength (W) of 3, an expectation (E) of10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc.Natl. Acad. Sci. USA 89, 10915 (1989))

For purposes of classifying amino acids substitutions as conservative ornonconservative, amino acids are grouped as follows: Group I(hydrophobic sidechains): norleucine, met, ala, val, leu, ile; Group II(neutral hydrophilic side chains): cys, ser, thr; Group III (acidic sidechains): asp, glu; Group IV (basic side chains): asn, gln, his, lys,arg; Group V (residues influencing chain orientation): gly, pro; andGroup VI (aromatic side chains): trp, tyr, phe. Conservativesubstitutions involve substitutions between amino acids in the sameclass. Non-conservative substitutions constitute exchanging a member ofone of these classes for a member of another.

The term “all-D” refers to peptides having ≧75%, ≧80%, ≧85%, ≧90%, ≧95%,and 100% D-configuration amino acids.

The term “agent” is used to describe a compound that has or may have apharmacological activity. Agents include compounds that are known drugs,compounds for which pharmacological activity has been identified butwhich are undergoing further therapeutic evaluation, and compounds thatare members of collections and libraries that are to be screened for apharmacological activity.

“Amyloid disease” or “amyloidosis” refers to any number of disorderswhich have as a symptom or as part of its pathology the accumulation orformation of amyloid plaques. An “amyloid plaque” is an extracellulardeposit composed mainly of proteinaceous fibrils. Generally, the fibrilsare composed of a dominant protein or peptide; however, the plaque mayalso include additional components that are peptide or non-peptidemolecules, as described herein.

An “amyloid protein” or “amyloid peptide” is a protein or peptidecapable of undergoing cleavage, conformational change, aggregation orfibrillogenesis, resulting in the formation of pathological oligomers,amyloid fibrils, amyloid plaques and/or amyloid components.

An “amyloid component” is any molecular entity that is present in anamyloid plaque including antigenic portions of such molecules. Amyloidcomponents include but are not limited to proteins, peptides,proteoglycans, and carbohydrates.

An “anti-amyloid agent” is an agent which is capable of producing animmune response against an amyloid plaque component in a vertebratesubject, when administered by active or passive immunization techniques.

An “AA protein” or “AA peptide” refers to the form of amyloid protein Aprotein or peptide formed by proteolytic cleavage of serum amyloid Aprotein (SAA), whether monomeric or aggregated, soluble or insoluble.

An “aggregated amyloid protein” or “aggregated amyloid peptide” or“amyloid aggregate” refers to a pathological, non-monomeric, aggregatedform of an amyloid protein or amyloid peptide. Aggregated amyloidproteins and amyloid peptides can be soluble or insoluble. Someaggregated amyloid proteins and aggregated amyloid peptides can formoligomers, fibrils and/or amyloid plaques. Examples of such aggregatedamyloid proteins and amyloid peptides, including fibril peptides andproteins are provided herein.

An “AA aggregate” refers to an aggregated form of AA.

Therapeutic agents of the invention are typically substantially purefrom undesired contaminant. This means that an agent is typically atleast about 50% w/w (weight/weight) purity, as well as beingsubstantially free from interfering proteins and contaminants. Sometimesthe agents are at least about 80% w/w and, more preferably at least 90or about 95% w/w purity. However, using conventional proteinpurification techniques, homogeneous peptides of at least 99% w/w can beobtained. Therapeutic agents of the invention may prevent, effectprophylaxis of, or treat a disease associated with amyloid deposits.

Specific binding between two entities means the entities have a mutualaffinity for each other that is at least 10-, 100- or 100-fold greaterthan the affinity of either entity for a control, such as unrelatedantigen or antibody to a different antigen. The mutual affinity of thetwo entities for each other is usually at least 10⁷ M⁻¹, 10⁸ M⁻¹, 10⁹M⁻¹, or 10¹⁰ M. Affinities greater than 10⁸ M⁻¹ are preferred.

The term “immunoglobulin” or “antibody” (used interchangeably herein)refers to an antigen-binding protein having a basic four-polypeptidechain structure consisting of two heavy and two light chains, saidchains being stabilized, for example, by interchain disulfide bonds,which has the ability to specifically bind antigen. Both heavy and lightchains are folded into domains. The term “domain” refers to a globularregion of a heavy or light chain polypeptide comprising peptide loops(e.g., comprising 3 to 4 peptide loops) stabilized, for example, byβ-pleated sheet and/or intrachain disulfide bond. Domains are furtherreferred to herein as “constant” or “variable”, based on the relativelack of sequence variation within the domains of various class membersin the case of a “constant” domain, or the significant variation withinthe domains of various class members in the case of a “variable” domain.“Constant” domains on the light chain are referred to interchangeably as“light chain constant regions”, “light chain constant domains”, “CL”regions or “CL” domains. “Constant” domains on the heavy chain arereferred to interchangeably as “heavy chain constant regions”, “heavychain constant domains”, “CH” regions or “CH” domains. “Variable”domains on the light chain are referred to interchangeably as “lightchain variable regions”, “light chain variable domains”, “VL” regions or“VL” domains. “Variable” domains on the heavy chain are referred tointerchangeably as “heavy chain constant regions”, “heavy chain constantdomains”, “CH” regions or “CH” domains.

The term “region” refers to a part or portion of an antibody chain andincludes constant or variable domains as defined herein, as well as morediscrete parts or portions of said domains. For example, light chainvariable domains or regions include “complementarity determiningregions” or “CDRs” interspersed among “framework regions” or “FRs”, asdefined herein.

Immunoglobulins or antibodies can exist in monomeric or polymeric form.The term “antigen-binding fragment” refers to a polypeptide fragment ofan immunoglobulin or antibody binds antigen or competes with intactantibody (i.e., with the intact antibody from which they were derived)for antigen binding (i.e., specific binding). The term “conformation”refers to the tertiary structure of a protein or polypeptide (e.g., anantibody, antibody chain, domain or region thereof). For example, thephrase “light (or heavy) chain conformation” refers to the tertiarystructure of a light (or heavy) chain variable region, and the phrase“antibody conformation” or “antibody fragment conformation” refers tothe tertiary structure of an antibody or fragment thereof.

“Specific binding” of an antibody mean that the antibody exhibitsappreciable affinity for antigen or a preferred epitope and, preferably,does not exhibit significant crossreactivity. “Appreciable” or preferredbinding include binding with an affinity of at least 10⁶, 10⁷, 10⁸, 10⁹M⁻¹, or 10¹⁰ M. Affinities greater than 10⁷ M⁻¹, preferably greater than10⁸ M⁻¹ are more preferred. Values intermediate of those set forthherein are also intended to be within the scope of the present inventionand a preferred binding affinity can be indicated as a range ofaffinities, for example, 10⁶ to 10¹⁰ M⁻¹, preferably 10⁷ to 10¹⁰ M⁻¹,more preferably 10⁸ to 10¹⁰ M. An antibody that “does not exhibitsignificant crossreactivity” is one that will not appreciably bind to anundesirable entity (e.g., an undesirable proteinaceous entity). Forexample, an antibody that specifically binds to AA will appreciably bindAA but will not significantly react with non-AA proteins or peptides(e.g., non-AA proteins or peptides included in plaques). An antibodyspecific for a preferred epitope will, for example, not significantlycrossreact with remote epitopes on the same protein or peptide. Specificbinding can be determined according to any art-recognized means fordetermining such binding. Preferably, specific binding is determinedaccording to Scatchard analysis and/or competitive binding assays.

Antigen-binding antibody fragments are produced by recombinant DNAtechniques, or by enzymatic or chemical cleavage of intactimmunoglobulins. Binding fragments include Fab, Fab′, F(ab′)₂, Fabc, Fv,single chains, and single-chain antibodies. Additional antibodyfragments and effector function variants are discussed herein in thesection entitled “Antibodies”. Other than “bispecific” or “bifunctional”immunoglobulins or antibodies, an immunoglobulin or antibody isunderstood to have each of its binding sites identical. A “bispecific”or “bifunctional antibody” is an artificial hybrid antibody having twodifferent heavy/light chain pairs and two different binding sites.Bispecific antibodies can be produced by a variety of methods includingfusion of hybridomas or linking of Fab′ fragments. See, e.g.,Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelnyet al., J. Immunol. 148, 1547-1553 (1992).

The term “humanized immunoglobulin” or “humanized antibody” refers to animmunoglobulin or antibody that includes at least one humanizedimmunoglobulin or antibody chain (i.e., at least one humanized light orheavy chain). The term “humanized immunoglobulin chain” or “humanizedantibody chain” (i.e., a “humanized immunoglobulin light chain” or“humanized immunoglobulin heavy chain”) refers to an immunoglobulin orantibody chain (i.e., a light or heavy chain, respectively) having avariable region that includes a variable framework region substantiallyfrom a human immunoglobulin or antibody and complementarity determiningregions (CDRs) (e.g., at least one CDR, preferably two CDRs, morepreferably three CDRs) substantially from a non-human immunoglobulin orantibody, and further includes constant regions (e.g., at least oneconstant region or portion thereof, in the case of a light chain, andpreferably three constant regions in the case of a heavy chain). Theterm “humanized variable region” (e.g., “humanized light chain variableregion” or “humanized heavy chain variable region”) refers to a variableregion that includes a variable framework region substantially from ahuman immunoglobulin or antibody and complementarity determining regions(CDRs) substantially from a non-human immunoglobulin or antibody.

The phrase “substantially from a human immunoglobulin or antibody” or“substantially human” means that, when aligned to a human immunoglobulinor antibody amino sequence for comparison purposes, the region shares atleast 80-90%, preferably 90-95%, more preferably 95-99% identity (i.e.,local sequence identity) with the human framework or constant regionsequence, allowing, for example, for conservative substitutions,consensus sequence substitutions, germline substitutions, backmutations,and the like. The introduction of conservative substitutions, consensussequence substitutions, germline substitutions, backmutations, and thelike, is often referred to as “optimization” of a humanized antibody orchain. The phrase “substantially from a non-human immunoglobulin orantibody” or “substantially non-human” means having an immunoglobulin orantibody sequence at least 80-95%, preferably 90-95%, more preferably,96%, 97%, 98%, or 99% identical to that of a non-human organism, e.g., anon-human mammal.

Accordingly, all regions or residues of a humanized immunoglobulin orantibody, or of a humanized immunoglobulin or antibody chain, exceptpossibly the CDRs, are substantially identical to the correspondingregions or residues of one or more native human immunoglobulinsequences. The term “corresponding region” or “corresponding residue”refers to a region or residue on a second amino acid or nucleotidesequence which occupies the same (i.e., equivalent) position as a regionor residue on a first amino acid or nucleotide sequence, when the firstand second sequences are optimally aligned for comparison purposes.

The terms “humanized immunoglobulin” or “humanized antibody” are notintended to encompass chimeric immunoglobulins or antibodies, as definedinfra. Although humanized immunoglobulins or antibodies are chimeric intheir construction (i.e., comprise regions from more than one species ofprotein), they include additional features (i.e., variable regionscomprising donor CDR residues and acceptor framework residues) not foundin chimeric immunoglobulins or antibodies, as defined herein.

The term “chimeric immunoglobulin” or antibody refers to animmunoglobulin or antibody whose variable regions derive from a firstspecies and whose constant regions derive from a second species.Chimeric immunoglobulins or antibodies can be constructed, for exampleby genetic engineering, from immunoglobulin gene segments belonging todifferent species.

An “antigen” is an entity (e.g., a protenaceous entity or peptide) towhich an antibody specifically binds.

The term “epitope” or “antigenic determinant” refers to a site on anantigen to which an immunoglobulin or antibody (or antigen bindingfragment thereof) specifically binds. Epitopes can be formed both fromcontiguous amino acids or noncontiguous amino acids juxtaposed bytertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatialconformation. Methods of determining spatial conformation of epitopesinclude, for example, x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols in Methods inMolecular Biology, Vol. 66, G. E. Morris, Ed. (1996).

Representative antibodies of the invention include an antibody orfragment thereof that specifically binds to an epitope that includesX₁EDX₂ in an aggregated amyloid protein, which binds to the epitopeincluding X₁EDX₂ that is also bound by e.g. a 2A4, 7D8, or 8G9 antibody.Antibodies that recognize the same epitope can be identified in a simpleimmunoassay showing the ability of one antibody to block the binding ofanother antibody to a target antigen, i.e., a competitive binding assay.Competitive binding is determined in an assay in which theimmunoglobulin under test inhibits specific binding of a referenceantibody to a common antigen, such as Aβ. Numerous types of competitivebinding assays are known, for example: solid phase direct or indirectradioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay (see Stahli et al.,Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidinEIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phasedirect labeled assay, solid phase direct labeled sandwich assay (seeHarlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborPress (1988)); solid phase direct label RIA using I-125 label (see Morelet al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidinEIA (Cheung et al., Virology 176:546 (1990)); and direct labeled RIA.(Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)). Typically, suchan assay involves the use of purified antigen bound to a solid surfaceor cells bearing either of these, an unlabeled test immunoglobulin and alabeled reference immunoglobulin. Competitive inhibition is measured bydetermining the amount of label bound to the solid surface or cells inthe presence of the test immunoglobulin. Usually the test immunoglobulinis present in excess. Usually, when a competing antibody is present inexcess, it will inhibit specific binding of a reference antibody to acommon antigen by at least 50-55%, 55-60%, 60-65%, 65-70% 70-75% ormore.

An epitope is also recognized by immunologic cells, for example, B cellsand/or T cells. Cellular recognition of an epitope can be determined byin vitro assays that measure antigen-dependent proliferation, asdetermined by ³H-thymidine incorporation, by cytokine secretion, byantibody secretion, or by antigen-dependent killing (cytotoxic Tlymphocyte assay).

The term “neoepitope” refers to a new and/or unique site on an antigento which B and/or T cells respond.

The term “neoepitope antibodies” refer to antibodies that specificallyrecognize a new N- or C-terminal amino acid sequence exposed byproteolytic cleavage of a molecule, but does not bind to such an epitopeon the native (uncleaved) molecule. The term “neoepitope antibodies” mayrefer to antibodies that specifically recognize a new N- or C-terminalamino acid sequence exposed by proteolytic cleavage of SAA, but do notbind to such an epitope on the native (uncleaved) SAA molecule. Someneoepitope antibodies bind to either soluble or insoluble AA and resultin dissociation of AA aggregates, including AA fibrils. A “neoepitopeantibody” may also be an antibody that specifically recognizes a newepitope that is only available to bind to an antibody after a proteinundergoes a conformation change, for example, as in the case of ALamyloidosis and light chain, when only the light chain is expressed andforms amyloid.

The term “immunological” or “immune” response is the development of abeneficial humoral (antibody mediated) and/or a cellular (mediated byantigen-specific T cells or their secretion products) response directedagainst an amyloid peptide in a recipient patient. Such a response canbe an active response induced by administration of immunogen or apassive response induced by administration of antibody or primedT-cells. A cellular immune response is elicited by the presentation ofpolypeptide epitopes in association with Class I or Class II MHCmolecules to activate antigen-specific CD4⁺ T helper cells and/or CD8⁺cytotoxic T cells. The response may also involve activation ofmonocytes, macrophages, NK cells, basophils, dendritic cells,astrocytes, microglia cells, eosinophils or other components of innateimmunity. The presence of a cell-mediated immunological response can bedetermined by proliferation assays (CD4⁺ T cells) or CTL (cytotoxic Tlymphocyte) assays (see Burke, supra; Tigges, supra). The relativecontributions of humoral and cellular responses to the protective ortherapeutic effect of an immunogen can be distinguished by separatelyisolating antibodies and T-cells from an immunized syngeneic animal andmeasuring protective or therapeutic effect in a second subject.

An “immunogenic agent” or “immunogen” is capable of inducing animmunological response against itself on administration to a mammal,optionally in conjunction with an adjuvant.

The term “naked polynucleotide” refers to a polynucleotide not complexedwith colloidal materials. Naked polynucleotides are sometimes cloned ina plasmid vector.

The term “adjuvant” refers to a compound that when administered inconjunction with an antigen augments the immune response to the antigen,but when administered alone does not generate an immune response to theantigen. Adjuvants can augment an immune response by several mechanismsincluding lymphocyte recruitment, stimulation of B and/or T cells, andstimulation of macrophages.

The term “effective dose” or “effective dosage” is defined as an amountsufficient to achieve or at least partially achieve the desired effect.The term “therapeutically effective dose” is defined as an amountsufficient to cure or at least partially arrest the disease and itscomplications in a patient already suffering from the disease. Amountseffective for this use will depend upon the severity of the infectionand the general state of the patient's own immune system.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

The invention provides antibodies or antigen-binding fragments thereofthat specifically bind to an epitope that includes X₁EDX₂ in anaggregated amyloid protein, and which competes for binding to theepitope comprising X₁EDX₂ with e.g., a 2A4, 7D8, or 8G9 antibody.Competition between antibodies is determined by an assay in which theimmunoglobulin under test inhibits specific binding of a referenceantibody to a common antigen, such as AA. Numerous types of competitivebinding assays are known, for example: solid phase direct or indirectradioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay (see Stahli et al.,Methods in Enzymology, 9:242-253 (1983)); solid phase directbiotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614-3619(1986)); solid phase direct labeled assay, solid phase direct labeledsandwich assay (see Harlow and Lane, “Antibodies, A Laboratory Manual,”Cold Spring Harbor Press (1988)); solid phase direct label RIA using1-125 label (see Morel et al., Molec. Immunol. 25(1):7-15 (1988)); solidphase direct biotin-avidin EIA (Cheung et al., Virology, 176:546-552(1990)); and direct labeled RIA (Moldenhauer et al., Scand. J. Immunol.,32:77-82 (1990)). Typically, such an assay involves the use of purifiedantigen bound to a solid surface or cells expressing the antigen, anunlabeled test immunoglobulin and a labeled reference immunoglobulin.Competitive inhibition is measured by determining the amount of labelbound to the solid surface or cells in the presence of the testimmunoglobulin. Usually the test immunoglobulin is present in excess.Antibodies identified by competition assay (competing antibodies)include antibodies binding to the same epitope as the reference antibodyand antibodies binding to an adjacent epitope sufficiently proximal tothe epitope bound by the reference antibody for steric hindrance tooccur. Usually, when a competing antibody is present in excess, it willinhibit specific binding of a reference antibody to a common antigen byat least 50% to 75%.

An antibody that specifically binds to an amyloid protein means anantibody that binds to the amyloid protein with an affinity of at least10⁷ M. Some antibodies bind to the amyloid protein with affinitiesbetween 10⁸ M⁻¹ and 10¹¹ M.

An antibody that specifically binds to aggregated amyloid protein suchas aggregated AA without specifically binding to monomeric amyloidprotein means an antibody that binds to aggregated amyloid protein, suchas, for example fibrils (e.g., AA in aggregated β-pleated sheet formsuch as from a cadaver of a former AA Amyloidosis patient or atransgenic animal model) as described above and has at least a ten foldand usually at least 100-fold lower specific binding affinity formonomeric forms of the amyloid protein. For example, such an antibodymight bind to soluble AA with an affinity of 10⁹ M⁻¹ and to plaques withan affinity less than 10⁷ M⁻¹. The affinity of such antibodies forplaques is usually less than 10⁷ or 10⁶ M⁻¹. Such antibodies areadditionally or alternatively defined by fluorescence intensity relativeto an irrelevant control antibody (e.g., an antibody or mixture ofpolyclonal antibodies to a reversemer AA peptide) when the antibodiesare contacted with fibrils and binding assessed by fluorescentlylabeling. The fluorescence intensity of antibodies that bind to solubleAA peptide without binding to plaques is within a factor of five,sometimes within a factor of two and sometimes indistinguishable withinexperimental error from that of the control antibody.

Compositions or methods “comprising” one or more recited elements mayinclude other elements not specifically recited. For example, acomposition that comprises AA peptide encompasses both an isolated AApeptide and AA peptide as a component of a larger polypeptide sequence.

II. Amyloid Diseases

1. Overview and Pathogenesis

Amyloid diseases or amyloidoses include a number of disease stateshaving a wide variety of outward symptoms. These disorders have incommon the presence of abnormal extracellular deposits of proteinfibrils, known as “amyloid deposits” or “amyloid plaques” that areusually about 10-100 μm in diameter and are localized to specific organsor tissue regions. Such plaques are composed primarily of a naturallyoccurring soluble protein or peptide. These insoluble deposits arecomposed of generally lateral aggregates of fibrils that areapproximately 10-15 nm in diameter. Amyloid fibrils produce acharacteristic apple green birefringence in polarized light, whenstained with Congo Red dye. The disorders are classified on the basis ofthe major fibril components forming the plaque deposits, as discussedbelow.

The peptides or proteins forming the plaque deposits are often producedfrom a larger precursor protein. More specifically, the pathogenesis ofamyloid fibril deposits generally involves proteolytic cleavage of an“abnormal” precursor protein into fragments. These fragments generallyaggregate into anti-parallel β pleated sheets; however, certainundegraded forms of precursor protein have been reported to aggregateand form fibrils in familial amyloid polyneuropathy (varianttransthyretin fibrils) and dialysis-related amyloidosis (β₂microglobulin fibrils) (Tan, et al., 1994, supra).

2. Clinical Syndromes

This section provides descriptions of major types of amyloidoses,including their characteristic plaque fibril compositions. It is ageneral discovery of the present invention that amyloid diseases can betreated by administering agents that serve to stimulate an immuneresponse against a component or components of the variousdisease-specific amyloid deposits. As discussed in more detail inSection C below, such components are preferably constituents of thefibrils that form the plaques. The sections below serve to exemplifymajor forms of amyloidosis and are not intended to limit the invention.

a. AL Amyloidoses

AL amyloid deposition is generally associated with almost any dyscrasiaof the B lymphocyte lineage, ranging from malignancy of plasma cells(multiple myeloma) to benign monoclonal gammopathy. At times, thepresence of amyloid deposits may be a primary indicator of theunderlying dyscrasia.

Fibrils of AL amyloid deposits are composed of monoclonal immunoglobulinlight chains or fragments thereof. More specifically, the fragments arederived from the N-terminal region of the light chain (kappa or lambda)and contain all or part of the variable (V_(L)) domain thereof. Depositsgenerally occur in the mesenchymal tissues, causing peripheral andautonomic neuropathy, carpal tunnel syndrome, macroglossia, restrictivecardiomyopathy, arthropathy of large joints, immune dyscrasias,myelomas, as well as occult dyscrasias. However, it should be noted thatalmost any tissue, particularly visceral organs such as the heart, maybe involved.

b. Hereditary Systemic Amyloidoses

There are many forms of hereditary systemic amyloidoses. Although theyare relatively rare conditions, adult onset of symptoms and theirinheritance patterns (usually autosomal dominant) lead to persistence ofsuch disorders in the general population. Generally, the syndromes areattributable to point mutations in the precursor protein leading toproduction of variant amyloidogenic peptides or proteins. Table 2summarizes the fibril composition of exemplary forms of these disorders.

TABLE 2 Hereditary Amyloidoses^(a) Fibril Peptide/Protein Geneticvariant Clinical Syndrome Transthyretin and fragments Met30, many othersFamilial amyloid (ATTR) polyneuropathy (FAP), (mainly peripheral nerves)Transthyretin and fragments Thr45, Ala60, Ser84, Cardiac involvement(ATTR) Met111, Ile122 predominant without neuropathy N-terminal fragmentof Arg 26 Familial amyloid Apolipoprotein A1 (apoAI) polyneuropathy(FAP), (mainly peripheral nerves) N-terminal fragment of Arg26, Arg50,Arg Ostertag-type, non-neuropathic Apolipoprotein A1 (AapoAI) 60, others(predominantly visceral involvement) Lysozyme (Alys) Thr56, His67Ostertag-type, non-neuropathic (predominantly visceral involvement)Fibrogen α chain fragment Leu554, Val 526 Ostertag-type, non-neuropathic(predominantly visceral involvement) Gelsolin fragment (Agel) Asn187,Tyr187 Cranial neuropathy with lattice corneal dystrophy Cystatin Cfragment Glu68 Hereditary cerebral hemorrhage (cerebral amyloidangiopathy)—Icelandic type β-amyloid protein (Aβ) Gln693 Hereditarycerebral derived from Amyloid hemorrhage (cerebral amyloid PrecursorProtein (APP) angiopathy)—Dutch type β-amyloid protein (Aβ) Ile717,Phe717, Familial Alzheimer's Disease derived from Amyloid Gly717Precursor Protein (APP) β-amyloid protein (Aβ) Asn670, Leu671 FamilialDementia—probable derived from Amyloid Alzheimer's Disease PrecursorProtein (APP) Prion Protein (PrP) derived Leu102, Val167, FamilalCreutzfeldt-Jakob from PrP precursor protein Asn178, Lys200 disease;Gerstmann-Sträussler- 51-91 insert Scheinker syndrome (hereditaryspongiform encephalopathies, prion diseases) AA derived from SerumFamilal Mediterranean fever, amyloid A protein (ApoSSA) predominantrenal involvement (autosomal recessive) AA derived from SerumMuckle-Well's syndrome, amyloid A protein (ApoSSA) nephropathy,deafness, urticaria, limb pain Unknown Cardiomyopathy with persistentatrial standstill Unknown Cutaneous deposits (bullous, papular,pustulodermal) ^(a)Data derived from Tan & Pepys, 1994, supra.

The data provided in Table 2 are exemplary and are not intended to limitthe scope of the invention. For example, more than 40 separate pointmutations in the transthyretin gene have been described, all of whichgive rise to clinically similar forms of familial amyloidpolyneuropathy.

Transthyretin (TTR) is a 14 kilodalton protein that is also sometimesreferred to as prealbumin. It is produced by the liver and choroidplexus, and it functions in transporting thyroid hormones and vitamin A.At least 50 variant forms of the protein, each characterized by a singleamino acid change, are responsible for various forms of familial amyloidpolyneuropathy. For example, substitution of proline for leucine atposition 55 results in a particularly progressive form of neuropathy;substitution of methionine for leucine at position 111 resulted in asevere cardiopathy in Danish patients. Amyloid deposits isolated fromheart tissue of patients with systemic amyloidosis have revealed thatthe deposits are composed of a heterogeneous mixture of TTR andfragments thereof, collectively referred to as ATTR, the full lengthsequences of which have been characterized. ATTR fibril components canbe extracted from such plaques and their structure and sequencedetermined according to the methods known in the art (e.g., Gustaysson,A., et al., Laboratory Invest. 73: 703-708, 1995; Kametani, F., et al.,Biochem. Biophys. Res. Commun. 125: 622-628, 1984; Pras, M., et al.,PNAS 80: 539-42, 1983).

Persons having point mutations in the molecule apolipoprotein AI (e.g.,Gly→Arg26; Trp→Arg50; Leu→Arg60) exhibit a form of amyloidosis(“Östertag type”) characterized by deposits of the proteinapolipoprotein AI or fragments thereof (AApoAI). These patients have lowlevels of high density lipoprotein (HDL) and present with a peripheralneuropathy or renal failure.

A mutation in the alpha chain of the enzyme lysozyme (e.g., Ile→Thr56 orAsp→His57) is the basis of another form of Östertag-type non-neuropathichereditary amyloid reported in English families. Here, fibrils of themutant lysozyme protein (Alys) are deposited, and patients generallyexhibit impaired renal function. This protein, unlike most of thefibril-forming proteins described herein, is usually present in whole(unfragmented) form (Benson, M. D., et al. CIBA Fdn. Symp. 199: 104-131,1996).

β-amyloid peptide (Aβ) is a 39-43 amino acid peptide derived byproteolysis from a large protein known as beta amyloid precursor protein(βAPP). Mutations in βAPP result in familial forms of Alzheimer'sdisease, Down's syndrome and/or senile dementia, characterized bycerebral deposition of plaques composed of Aβ fibrils and othercomponents, which are described in further detail below. Known mutationsin APP associated with Alzheimer's disease occur proximate to thecleavage sites of β or γ secretase, or within Aβ. For example, position717 is proximate to the site of γ-secretase cleavage of APP in itsprocessing to Aβ, and positions 670/671 are proximate to the site ofβ-secretase cleavage. Mutations at any of these residues may result inAlzheimer's disease, presumably by causing an increase the amount of the42/43 amino acid form of Aβ generated from APP. The structure andsequence of Aβ peptides of various lengths are well known in the art.Such peptides can be made according to methods known in the art (e.g.,Glenner and Wong, Biochem Biophys. Res. Comm. 129: 885-890, 1984;Glenner and Wong, Biochem Biophys. Res. Comm. 122: 1131-1135, 1984). Inaddition, various forms of the peptides are commercially available.

Synuclein is a synapse-associated protein that resembles an alipoproteinand is abundant in neuronal cytosol and presynaptic terminals. A peptidefragment derived from α-synuclein, termed NAC, is also a component ofamyloid plaques of Alzheimer's disease. (Clayton, et al., 1998). Thiscomponent also serves as a target for immunologically-based treatmentsof the present invention, as detailed below.

Gelsolin is a calcium binding protein that binds to and fragments actinfilaments. Mutations at position 187 (e.g., Asp→Asn; Asp→Tyr) of theprotein result in a form of hereditary systemic amyloidosis, usuallyfound in patients from Finland, as well as persons of Dutch or Japaneseorigin. In afflicted individuals, fibrils formed from gelsolin fragments(Agel), usually consist of amino acids 173-243 (68 kDa carboxyterminalfragment) and are deposited in blood vessels and basement membranes,resulting in corneal dystrophy and cranial neuropathy which progressesto peripheral neuropathy, dystrophic skin changes and deposition inother organs. (Kangas, H., et al. Human Mol. Genet. 5(9): 1237-1243,1996).

Other mutated proteins, such as mutant alpha chain of fibrinogen (AfibA)and mutant cystatin C (Acys) also form fibrils and producecharacteristic hereditary disorders. AfibA fibrils form depositscharacteristic of a normeuropathic hereditary amyloid with renaldisease; Acys deposits are characteristic of a hereditary cerebralamyloid angiopathy reported in Iceland. (Isselbacher, et al., Harrison'sPrinciples of Internal Medicine, McGraw-Hill, San Francisco, 1995;Benson, et al., supra.). In at least some cases, patients with cerebralamyloid angiopathy (CAA) have been shown to have amyloid fibrilscontaining a non-mutant form of cystatin C in conjunction with betaprotein. (Nagai, A., et al. Molec. Chem. Neuropathol. 33: 63-78, 1998).

Certain forms of prion disease are now considered to be heritable,accounting for up to 15% of cases, which were previously thought to bepredominantly infectious in nature. (Baldwin, et al., in ResearchAdvances in Alzheimer's Disease and Related Disorders, John Wiley andSons, New York, 1995). In such prion disorders, patients develop plaquescomposed of abnormal isoforms of the normal prion protein (PrP^(c)). Apredominant mutant isoform, PrP^(Sc), also referred to as AScr, differsfrom the normal cellular protein in its resistance to proteasedegradation, insolubility after detergent extraction, deposition insecondary lysosomes, post-translational synthesis, and high β-pleatedsheet content. Genetic linkage has been established for at least fivemutations resulting in Creutzfeldt-Jacob disease (CJD),Gerstmann-Sträussler-Scheinker syndrome (GSS), and fatal familialinsomnia (FFI). (Baldwin) Methods for extracting fibril peptides fromscrapie fibrils, determining sequences and making such peptides areknown in the art. (e.g., Beekes, M., et al. J. Gen. Virol. 76: 2567-76,1995).

For example, one form of GSS has been linked to a PrP mutation at codon102, while telencephalic GSS segregates with a mutation at codon 117.Mutations at codons 198 and 217 result in a form of GSS in whichneuritic plaques characteristic of Alzheimer's disease contain PrPinstead of Aβ peptide. Certain forms of familial CJD have beenassociated with mutations at codons 200 and 210; mutations at codons 129and 178 have been found in both familial CJD and FFI. (Baldwin, supra).

c. Senile Systemic Amyloidosis

Amyloid deposition, either systemic or focal, increases with age. Forexample, fibrils of wild type transthyretin (TTR) are commonly found inthe heart tissue of elderly individuals. These may be asymptomatic,clinically silent, or may result in heart failure. Asymptomaticfibrillar focal deposits may also occur in the brain (Aβ), corporaamylacea of the prostate (Aβ₂ microglobulin), joints and seminalvesicles.

d. Cerebral Amyloidosis

Local deposition of amyloid is common in the brain, particularly inelderly individuals. The most frequent type of amyloid in the brain iscomposed primarily of Aβ peptide fibrils, resulting in dementia orsporadic (non-hereditary) Alzheimer's disease. In fact, the incidence ofsporadic Alzheimer's disease greatly exceeds forms shown to behereditary. Fibril peptides forming these plaques are very similar tothose described above, with reference to hereditary forms of Alzheimer'sdisease (AD).

e. Dialysis-related Amyloidosis

Plaques composed of β₂ microglobulin (Aβ₂M) fibrils commonly develop inpatients receiving long term hemodialysis or peritoneal dialysis. β₂microglobulin is a 11.8 kilodalton polypeptide and is the light chain ofClass I MHC antigens, which are present on all nucleated cells. Undernormal circumstances, it is continuously shed from cell membranes and isnormally filtered by the kidney. Failure of clearance, such as in thecase of impaired renal function, leads to deposition in the kidney andother sites (primarily in collagen-rich tissues of the joints). Unlikeother fibril proteins, Aβ₂M molecules are generally present inunfragmented form in the fibrils. (Benson, supra).

f. Hormone-Derived Amyloidoses

Endocrine organs may harbor amyloid deposits, particularly in agedindividuals. Hormone-secreting tumors may also contain hormone-derivedamyloid plaques, the fibrils of which are made up of polypeptidehormones such as calcitonin (medullary carcinoma of the thyroid), isletamyloid polypeptide (amylin; occurring in most patients with Type IIdiabetes), and atrial natriuretic peptide (isolated atrial amyloidosis).sequences and structures of these proteins are well known in the art.

g. Miscellaneous Amyloidoses

There are a variety of other forms of amyloid disease that are normallymanifest as localized deposits of amyloid. In general, these diseasesare probably the result of the localized production and/or lack ofcatabolism of specific fibril precursors or a predisposition of aparticular tissue (such as the joint) for fibril deposition. Examples ofsuch idiopathic deposition include nodular AL amyloid, cutaneousamyloid, endocrine amyloid, and tumor-related amyloid.

III. AA Amyloid Diseases

AA amyloidosis, formerly called secondary or reactive amyloidosisbecause it develops secondary to a preexisting or coexisting disease.Such diseases include, but are not limited to inflammatory diseases,such as rheumatoid arthritis, juvenile chronic arthritis, ankylosingspondylitis, psoriasis, psoriatic arthropathy, Reiter's syndrome, AdultStill's disease, Behcet's syndrome, and Crohn's disease. AA deposits arealso produced as a result of chronic microbial infections, such asleprosy, tuberculosis, bronchiectasis, decubitus ulcers, chronicpyelonephritis, osteomyelitis, and Whipple's disease. Certain malignantneoplasms can also result in AA fibril amyloid deposits. These includesuch conditions as Hodgkin's lymphoma, renal carcinoma, carcinomas ofgut, lung and urogenital tract, basal cell carcinoma, and hairy cellleukemia. AA amyloid disease may also result from inherited inflammatorydiseases such as Familial Mediterranean Fever. Additionally, AA amyloiddisease may result from lymphoproliferative disorders such asCastleman's Disease.

1. Inflammatory Diseases Associated with AA Amyloidosis

Rheumatoid arthritis is a chronic systemic disease primarily of thejoints. The symptoms of rheumatoid arthritis are marked by inflammatorychanges in the synovial membranes and articular structures (joints) andby atrophy and rarefaction (bone density decreases) of the bones. Inlate stages of rheumatoid arthritis, deformity and ankylosis (immobilityof the joint) develop. A model of rheumatoid arthritis can be induced inmice or rats by administering type II collagen in complete Freund'sadjuvant.

Juvenile chronic arthritis comes in many forms; the most common beingjuvenile rheumatoid arthritis. It can occur in children at any age, butfirst appears more commonly between the ages of 2 and 6 years. There are3 main types of juvenile rheumatoid arthritis, namely, pauci-articulararthritis, polyarticular arthritis, and systemic arthritis (also knownas Still's disease). Pauci-articular arthritis typically affects 4 orfewer joints, usually the larger ones such as the knees. It can beaccompanied by stiffness, causing the child to limp. Polyarticulararthritis is characterized by 5 or more joints being affected, mostcommonly the smaller joints in the hands and feet. Children withpolyarticular arthritis often have a more severe form of the disease.Systemic arthritis is characterized by joint swelling in combinationwith fever and a pink rash. The joints may not start to swell until somemonths or years after the fevers begin. It may also affect internalorgans such as the liver, heart, spleen and lymph nodes, and anemia iscommon. While systemic arthritis tends to abate of its own accord, asmall percentage of these children can have severe arthritis thatcontinues into adulthood.

Ankylosing spondylitis is a rheumatic disease that causes arthritis ofthe spine and sacroiliac joints and can cause inflammation of the eyes,lungs, and heart valves. It varies from intermittent episodes of backpain that occur throughout life to a severe chronic disease that attacksthe spine, peripheral joints and other body organs, resulting in severejoint and back stiffness, loss of motion and deformity as lifeprogresses.

Psoriasis is a common chronic, squamous dermatosis, marked byexacerbation and remissions and having a polygenic inheritance pattern.The symptoms of psoriasis are marked by the presence of rounded, dryscaling patches of various sizes, covered by a grayish white or silverywhite scales that have a predilection for the extensor surfaces, nails,scalp, genitalia and the lumbosacral region.

Psoriatic arthropathy is a disorder in which psoriasis is linked to thedevelopment of arthritis. The disorder can be exhibited in a variety ofways. The arthritis is generally mild and involves only a few joints. Ina few patients, the disease is severe and usually affects the fingersand the spine. When the spine is affected, the symptoms are very muchlike those of ankylosing spondylitis.

Reiter's syndrome is a group of symptoms consisting of arthritis,urethritis (inflammation of the urogenital tract), conjunctivitis(inflammation of the lining of the eye), and lesions of the skin andmucous membranes. Reiter's syndrome is also referred to as reactivearthritis, which means that the arthritis occurs as a “reaction” to aninfection that started elsewhere in the body. Chlamydia trachomatis isthe bacteria most often associated with Reiter's syndrome acquiredthrough sexual contact. Several different bacteria are associated withReiter's syndrome acquired through the digestive tract, includingSalmonella, Shigella, Yersinia, and Campylobacter.

Adult Still's disease, also called Adult Onset Still's Disease is a rareinflammatory condition that attacks internal organs, joints and otherparts of the body. It can appear and disappear suddenly. In very severecases, adult Still's disease becomes chronic and extremely debilitating,causing terrible pain and stiffness. After many years, the diseasecripples vital organs such as the heart and lungs.

Behcet's syndrome is a multisystem disorder presenting with recurrentoral and/or genital ulcerations, chronic relapsing uveitis that maycause blindness and neurologic impairments. It is characterized by 4major symptoms: oral aphthous ulcers, skin lesions, ocular symptoms, andgenital ulcerations, and occasionally by inflammation in tissues andorgans throughout the body, including the gastrointestinal tract,central nervous system, vascular system, lungs, and kidneys. Thearthritis of Behcet's syndrome is usually intermittent, self-limited,not deforming and localized to the knees and ankles.

Crohn's disease is a chronic granulomatous (small grain-like body orgrowth) inflammatory disease involving any part of the gastrointestinaltract from the mouth to anus; but commonly involving the ileum (lowerthree-fifths of the small intestines) with scarring and thickening ofthe bowel wall. The symptoms of Crohn's disease include the presence ofchronic diarrhea, increased bowel sounds, cramping, possibly evidencedby weight loss and aversion to eating.

2. Chronic Microbial Infection Diseases Associated with AA Amyloidosis

Leprosy is an infectious disease characterized by disfiguring skinsores, peripheral nerve damage, and progressive debilitation. Leprosy iscaused by the organism Mycobacterium leprae, which is not verycontagious and has a long incubation period. Leprosy has two commonforms, tuberculoid and lepromatous. Both forms produce sores on theskin, but the lepromatous form is most severe, producing large,disfiguring nodules (lumps and bumps). Leptosy eventually causesperipheral neurological damage. Patients with long-term leprosy may losethe use of their hands or feet due to repeated injury resulting fromlack of sensation.

Tuberculosis is a contagious bacterial infection caused by Mycobacteriumtuberculosis. The disease is characterized by the development ofgranulomas (granular tumors) in the infected tissues. The lungs areprimarily involved, but the infection can spread to other organs.Bronchiectasis is an abnormal destruction and dilation of the largeairways.

Bronchiectasis is often caused by recurrent inflammation or infection ofthe airways. A classic bacterium that is seen in patients withbronchiectasis is Pseudomonas aeruginosa, which is notoriously hard toeradicate. Repeated infections of the airways by this bacterium can leadto colonization of the bronchi by this organism which predisposes suchpeople to Pseudomonal pneumonias, which requires special antibiotics totreat.

Decubitus ulcer also known as pressure ulcer or bedsore is an ulcerationof the skin and underlying tissues caused by prolonged pressure over theaffected area. They start as reddened skin but gets progressively worse,forming a blister, then an open sore, and finally a crater. Theseulcerations usually occur over bony prominences such as heels, coccyxarea of the buttock and the back of the head.

Chronic pyelonephritis is an infection of the kidney and the ureters(ducts that carry urine away from the kidney). Pyelonephritis most oftenoccurs as a result of urinary tract infection, particularly in thepresence of occasional or persistent backflow of urine from the bladderinto the ureters or kidney pelvis.

Osteomyelitis is an acute or chronic bone infection, usually caused bybacteria. Often the infection initiates in another part of the body andspreads to the bone via the blood. When the bone is infected, pus isproduced within the bone, which may result in an abscess. The abscessthen deprives the bone of its blood supply. Chronic osteomyelitisresults when bone tissue dies as a result of the lost blood supply.Chronic infection can persist intermittently for years.

Whipple's disease is a rare condition that causes inadequate absorptionof nutrients from the intestinal tract due to infection of theintestine. It is caused by the bacteria, Tropheryma whippelii. Symptomsinclude diarrhea, intestinal bleeding, abdominal pain, loss of appetite,weight loss, fatigue, and weakness. Arthritis and fever often occurseveral years before intestinal symptoms develop. Patients mayexperience neurological symptoms as well. Diagnosis is based on symptomsand the results of a biopsy of tissue from the small intestine or otherorgans that are affected. When recognized and treated, Whipple's diseasecan usually be cured. Without treatment, the condition is usually fatal.

3. Malignant Neoplasms Associated with AA Amyloidosis

Hodgkin's lymphoma is a cancer of lymphatic tissue found in the lymphnodes, spleen, liver, and bone marrow. The first sign of this cancer isoften an enlarged lymph node. The disease can spread to nearby lymphnodes and later may spread to the lungs, liver, or bone marrow.

Renal carcinoma is cancer of the kidney. The cancerous cells are foundin the lining of tubules in the kidney. The first symptom is usuallyblood in the urine. Sometimes both kidneys are involved. The cancerspreads easily, most often to the lungs and other organs. Renal cellcarcinoma is the most common type of kidney cancer followed by papillaryrenal cell carcinoma, chromophobe renal carcinoma and collecting ductrenal carcinoma. About 5% of renal carcinoma are unclassified becausetheir appearance doesn't fit into any of the other categories.

Carcinomas of the gut include gastrointestinal cancers such ascolorectal, pancreatic, stomach and esophageal. Colorectal cancer iscancer that starts in the large intestine or the rectum. Almost allcolorectal cancers begin as benign polyps which, over a period of manyyears, develop into cancers. Most cases of colorectal cancer have nosymptoms. Pancreatic cancer is a malignancy of the pancreas. Symptomsinclude abdominal pain, loss of appetite, significant weight loss andpainless jaundice. Stomach cancer, also called gastric cancer, candevelop in any part of the stomach and may spread throughout the stomachand to other organs; particularly the esophagus and the small intestine.It may also spread, through the stomach wall, to nearby lymph nodes andorgans such as the liver, pancreas, and the lungs, or to distant organssuch as the lymph nodes above the collar bone, the colon, and theovaries. Stomach cancer is often asymptomatic. Esophageal cancer ismalignancy of the esophagus. Symptoms include dysphagia (difficultyswallowing), pain and substantial weight loss.

Carcinomas of the lung are a cancer of the lungs characterized by thepresence of malignant tumours. There are two main types of lung cancer:non-small cell lung cancer and small cell lung cancer. Symptoms dependon the specific type of cancer, but may include chronic cough, coughingup blood, shortness of breath, wheezing, chest pain, loss of appetite,weight loss and fatigue.

Carcinomas of the urogenital tract include but are not limited toprostate cancer, bladder cancer, endometrial cancer, cervical cancer andovarian cancer. Prostate cancer involves a malignant tumor growth withinthe prostate gland. Symptoms may include frequent urination, difficultystarting and maintaining a steady stream of urine, blood in the urine,painful urination, difficulty achieving erection or painful ejaculation.Bladder cancer refers to any of several types of malignant growths ofthe urinary bladder. Symptoms include blood in the urine, frequenturination, painful urination, and urinary urgency. Endometrial cancerinvolves cancerous growth of the endometrium (lining of the uterus). Itmainly occurs after menopause, and presents with vaginal bleeding.Cervical cancer is a malignancy of the cervix. The early stages ofcervical cancer may be completely asymptomatic. Vaginal bleeding mayindicate the presence of malignancy. In advanced stages, metastases maybe present in the abdomen, lungs or elsewhere. Ovarian cancer is amalignant neoplasm of the ovaries. Ovarian cancer symptoms are oftenvague and non-specific, which include vague lower abdominal discomfort,sense of pelvic heaviness, abnormal menstrual cycle, vaginal bleeding,weight gain or loss, nonspecific gastrointestinal symptoms. Ovariancancers shed cancer cells that often implant on the uterus, bladder,bowel, and lining of the bowel wall. These cancer cells can beginforming new tumor growths before cancer is even suspected.

Basal cell carcinoma is a slow-growing skin tumor involving cancerouschanges in basal skin cells. Symptoms include skin lesions located onthe face, ear, neck, chest, back, or scalp; visible blood vessels in thelesion or adjacent skin; and persistent, non-healing sores. This cancerusually remains local and almost never spreads to distant parts of thebody, but it may continue to grow and invade nearby tissues andstructures, including the nerves, bones, and brain.

Hairy cell leukemia is a cancer of lymphocytes (B cells) that leads tolow blood counts. The disease is caused by the abnormally shaped B cellswith hair-like projections. Symptoms are often vague. The low bloodcounts caused by hairy cell leukemia can lead to infections, fatigue,and excessive bleeding.

4. Inherited Inflammatory Disease Associated with AA

Familial Mediterranean Fever is an inherited disorder characterized byrecurrent fever and inflammation, often involving the abdomen or thelung. Symptoms include inflammation in the lining of the abdominalcavity, chest cavity, skin, or joints occurs, along with high feversthat usually peak in 12 to 24 hours. Attacks may vary in severity ofsymptoms, and people are usually symptom free between attacks. Thisdisease is very rare. Risk factors include a family history of familialMediterranean Fever or having Mediterranean ancestry.

5. Lymphoproliferative Disorders Associated with AA Amyloidosis

Castleman's Disease is a form of lympoproliferative disordercharacterized pathologically by the presence of giant lymp nodehyperplasia with plasma cell infiltration. Patients with Castleman'sDisease commonly have fever, anemia, hypergammaglobulinaemia, and anincrease in the serum concentrations of acute phase reactant proteins,all of which are ascribed to the large amount of IL-6 produced in thelymph nodes.

IV. Serum Amyloid A

1. Human Serum Amyloid A

Serum amyloid A (SAA) is the circulating precursor of amyloid A protein,the fibrillar component of amyloid deposits. The structural studiesshowed that the human SAA is heterogeneous and represents a family ofpolymorphic SAA genes and protein products. The SAA gene superfamilycomprises a cluster of closely linked genes localized to 11p15.1. SeeSellar, G C et al. Genomics 19: 221-227 (1994). Four SAA genes have beendescribed in humans. Representative amino acid sequences of proteinsencoded by the four SAA genes are illustrated by FIG. 1. Two genes (SAA1and SAA2) encode acute-phase serum amyloid A (A-SAA) and arecoordinately induced in response to inflammation. SAA1 and SAA2 share95% sequence identity in both coding and noncoding regions. There arealpha, beta and gamma isoforms of human SAA1 and alpha and beta isoformsof human SAA2 as illustrated by FIGS. 18 and 19. SAA3 is a pseudogene.SAA4 encodes constitutive SAA and is minimally inducible. See Cunnane G.Bailliere's Clin. Rheumatol. 13(4): 615-628. All human SAA/AA moleculescontains a theoretical calcium-binding tetrapeptide sequence,Gly-Pro-Gly-Gly, of possible importance for self aggregation and withextrafibrillar moieties of amyloid in fibrillogenesis. See Fykse, E. M.et al. Biochem. J. 256:973-980 (1988) and Turnell et al. Mol. Biol. Med.3:387-407 (1986). The N terminal portion of SAA/AA is stronglyhydrophobic, probably of importance for self aggregation and othercomponents in amyloid deposits. See Husby et al. Clin. Immunol.Immunopathol. 70(1):2-9 (1994). The sequence of each isoform of AA andits relationship to its corresponding SAA isoform is illustrated byFIGS. 2-5. For example, human SAA1 alpha isoform has the sequence:

(SEQ ID NO: 1)H₂N-Met-Lys-Leu-Leu-Thr-Gly-Leu-Val-Phe-Cys-Ser-Leu-Val-Leu-Gly-Val-Ser-Ser-Arg-Ser-Phe-Phe-Ser-Phe-Leu-Gly-Glu-Ala-Phe-Asp-Gly-Ala-Arg-Asp-Met-Try-Arg-Ala-Tyr-Ser-Asp-Met-Arg-Glu-Ala-Asn-Tyr-Ile-Gly-Ser-Asp-Lys-Tyr-Phe-His-Ala-Arg-Gly-Asn-Tyr-Asp-Ala-Ala-Lys-Arg-Gly-Pro-Gly-Gly-Ala-Try-Ala-Ala-Glu-Val-Ile-Ser-Asp-Ala-Arg-Glu-Asn-Ile-Gln-Arg-Phe-Phe-Gly-His-Gly-Ala-Glu-Asp-Ser-Leu-Ala-Asp-Gln-Ala-Ala-Asn-Glu-Try-Gly-Arg-Ser-Gly-Lys-Asp-Pro-Asn-His-Phe-Arg-Pro-Ala-Gly-Leu-Pro-Glu-Lys-Tyr-OH.

AA, which is a proteolytic fragment of SAA, is also heterogeneous. Thepredominant human AA peptide consists of 76 amino acids. An example ofAA has the sequence:

(SEQ ID NO: 2)H₂N-Arg-Ser-Phe-Phe-Ser-Phe-Leu-Gly-Glu-Ala-Phe-Asp-Gly-Ala-Arg-Asp-Met-Try-Arg-Ala-Tyr-Ser-Asp-Met-Arg-Glu-Ala-Asn-Tyr-Ile-Gly-Ser-Asp-Lys-Tyr-Phe-His-Ala-Arg-Gly-Asn-Tyr-Asp-Ala-Ala-Lys-Arg-Gly-Pro-Gly-Gly-Ala-Try-Ala-Ala-Glu-Val-Ile-Ser-Asp-Ala-Arg-Glu-Asn-Ile-Gln-Arg-Phe-Phe-Gly-His-Gly-Ala-Glu-Asp-Ser-OH.

AA70-76 refers to an AA fragment beginning at residue 70 and ending atresidue 76 of (SEQ ID NO:2) consisting of the sequence GHGAEDS, (SEQ IDNO: 4), or corresponding segment from another naturally occurring AAprotein from a human or other species when the sequence of that proteinis maximally aligned with SEQ ID NO:2.

2. Murine Serum Amyloid A

In the mouse, four SAA genes have been described. Representative aminoacid sequences of proteins encoded by the four murine SAA genes areillustrated by FIG. 8. Mouse SAA gene family comprises four members thatare closely linked in the chromosome 7. Two of these genes encodingmajor mouse SAA isotypes (SAM and SAA2) share high sequence identity notonly in exons but also in introns and flanking regions and are inducedin approximately equal quantities in response to amyloid inductionmodels. These two isotypes differ in only 9 of 103 amino acid residues;however, only SAA2 is selectively deposited into amyloid fibrils. See deBeer M. C. Biochem J. 1991 280(Pt 1): 45-49 (1991); Hoffman J. S. et al.J Exp Med. 159:641-646 (1984); Shiroo M et al. Scand J. Immunol.26:709-716 (1987). SAA3 is a minor HDL apolipoprotein and peripherallyproduced acute phase. SAA4 is a constitutive subfamily that is a minornormal HDL apolipoprotein comprising more than 90% of the SAA duringhomeostasis. See Stearman R. S. et al. Nucleic Acids Research,14(2)797-809 (1986) and de Beer M. C. Genomics, 34(1):139-42 (1996).

Murine AA which is a proteolytic fragment of SAA is also heterogeneous.The sequence of each murine isoform of AA and its relationship to itscorresponding SAA isoform is illustrated by FIGS. 9-12. A sequencealignment of murine AA1, AA2, AA3 and AA4 is illustrated by FIG. 13.

Murine AA1 is the murine equivalent of human AA1. See FIG. 16. Inparticular, residues 69-75 of murine AA1 (GRGHEDT, SEQ ID NO: 9) aremaximally aligned with residues 70-76 of human AA1 (GHGAEDS, SEQ ID NO:4). See also FIG. 17.

3. Shar Pei Serum Amyloid A

The Shar Pei sequence is indicated in FIG. 20. Interestingly, thehomologous region in the human SAA protein -AEDS, (SEQ ID NO: 13)contains a conserved Thr to Ser substitution at position 76, as well assignificantly different side chain of the residue at position 73 (His toAla; FIG. 1). The -AEDS, (SEQ ID NO: 13), sequence is also observed inthe Shar Pei species of dog, a breed that is particularly susceptible toAA-amyloidosis and could provide a naturally occurring model of systemicAA in which to evaluate novel diagnostic and therapeutic applications ofAA amyloid-specific antibodies and other compounds.

4. The N-Terminal Segment of AA Protein Determines its FibrillogenicProperty

The amyloid fibril protein AA consists of a varying long N-terminal partof the precursor protein serum AA. Evidence shows that the amyloidogenicpart of the molecule is the N-terminal 10-15 amino acid long segment.Amino acid substitutions in this part of the molecule may explain whyonly one of the two mouse SAA isoforms is amyloidogenic. See WestermarkG. T. Biochem Biophys Res Commun. 182(1):27-33 (1992).

V. Other Human Amyloidogenic Proteins

The Genbank Accession Numbers and X₁EDX₂ sequences are provided below inTable 3 for several human amyloidogenic proteins, including some ofthose listed above in Table 2.

TABLE 3 Human Amyloidogenic Proteins GenBank Human amyloidogenic proteinConsensus sequence Accession Number SAA1 AEDS, (SEQ ID NO: 13) SAA2AEDS, (SEQ ID NO: 13) SAA3 AEDS, (SEQ ID NO: 13) SAA4 AEDS, (SEQ ID NO:13) anti-Sm immunoglobulin kappa light chain V AEDV, (SEQ ID NO: 23)AAB26897 region; monoclonal antibody 4B4 kappa chain immunoglobulinvariable region used by the PEDS, (SEQ ID NO: 26) AAC61608 ITC52 kappalight chain (subgroup V kappa III immunoglobulin variable region used bythe AEDV, (SEQ ID NO: 23) AAC61606 ITC48 kappa light chain (subgroup Vkappa IV) anti-RhD monoclonal T125 kappa light chain SEDF, (SEQ ID NO:24) AAW82027 precursor immunoglobulin kappa light chain precursor AEDV,(SEQ ID NO: 23) CAA45496 immunoglobulin kappa light chain variable PEDF,(SEQ ID NO: 22) AAT44350 region immunoglobulin kappa light chainvariable PEDF, (SEQ ID NO: 22) AAT44349 region immunoglobulin kappalight chain variable PEDF, (SEQ ID NO: 22) AAT44348 regionimmunoglobulin kappa light chain PEDF, (SEQ ID NO: 22) CAA09185immunoglobulin kappa light chain SEDF, (SEQ ID NO: 24) CAA09181immunoglobulin kappa light chain variable SEDF, (SEQ ID NO: 24) AAU14891region anti-rabies SOJA immunoglobulin kappa PEDF, (SEQ ID NO: 22)AAO17825 light chain anti-streptococcal/anti-myosin SEDF, (SEQ ID NO:24) AAB68786 immunoglobulin kappa light chain variable regionanti-streptococcal/anti-myosin PEDF, (SEQ ID NO: 22) AAB68785immunoglobulin kappa light chain variable regionanti-HLA-A2/anti-HLA-A28 PEDF, (SEQ ID NO: 22) AAC99644 immunoglobulinkappa light chain variable region immunoglobulin kappa light chain Vregion; PEDF, (SEQ ID NO: 22) AAB62946 anti-DNA antibody 18/2immunoglobulin kappa light chain PEDF, (SEQ ID NO: 22) BAF75949anti-HIV-1 gp120 immunoglobulin 48d PEDF, (SEQ ID NO: 22) AAR88370 kappalight chain immunoglobulin kappa light chain PEDL, (SEQ ID NO: 27)BAA97671 anti-Entamoeba histolytica immunoglobulin PEDF, (SEQ ID NO: 22)BAA82105 kappa light chain anti-Entamoeba histolytica immunoglobulinTEDV, (SEQ ID NO: 28) BAA82102 kappa light chain immunoglobulin kappalight chain PEDF, (SEQ ID NO: 22) AAC41705 anti-GM2 glanglioside IgMmonoclonal AEDV, (SEQ ID NO: 23) AAC26480 kappa light chain variableregion anti-SARS-CoV immunoglobulin kappa light PEDV, (SEQ ID NO: 151)AAT51719 chain variable region anti-SARS-CoV immunoglobulin kappa lightPEDF, (SEQ ID NO: 22) AAT51718 chain variable region immunoglobulinkappa light chain VLJ PEDF, (SEQ ID NO: 22) BAD27502 regionimmunoglobulin kappa light chain VLJ SEDF, (SEQ ID NO: 24) BAD27497region anti-HIV-1 gp120 immunoglobulin 47e PEDF, (SEQ ID NO: 22)AAR88378 kappa light chain anti-HIV-1 gp120 immunoglobulin 16c PEDF,(SEQ ID NO: 22) AAR88374 kappa light chain anti-HIV-1 gp120immunoglobulin 411g SEDF, (SEQ ID NO: 24) AAR88372 kappa light chainimmunoglobulin kappa light chain variable PEDF, (SEQ ID NO: 22) AAF14212region immunoglobulin kappa light chain variable PEDF, (SEQ ID NO: 22)AAF14211 region immunoglobulin kappa light chain variable PEDF, (SEQ IDNO: 22) AAF14210 region immunoglobulin kappa light chain variable PEDF,(SEQ ID NO: 22) AAF14209 region immunoglobulin V-region kappa lightchain PEDI, (SEQ ID NO: 21) AAR02415 immunoglobulin kappa light chainPEDF, (SEQ ID NO: 22) AAM46647 immunoglobulin kappa light chain AEDV,(SEQ ID NO: 23) AAM46643 anti-Entamoeba histolytica immunoglobulin PEDF,(SEQ ID NO: 22) BAA82103 kappa light chain immunoglobulin light chainkappa variable AEDV, (SEQ ID NO: 23) AAL65723 region immunoglobulinlight chain kappa variable PEDF, (SEQ ID NO: 22) AAL65718 regionimmunoglobulin light chain kappa variable SEDF, (SEQ ID NO: 24) AAL65717region immunoglobulin light chain kappa variable SEDF, (SEQ ID NO: 24)AAL65716 region immunoglobulin light chain kappa variable PEDF, (SEQ IDNO: 22) AAL65714 region immunoglobulin light chain kappa variable PEDF,(SEQ ID NO: 22) AAL65713 region immunoglobulin light chain kappavariable PEDF, (SEQ ID NO: 22) AAL65712 region immunoglobulin lightchain kappa variable PEDF, (SEQ ID NO: 22) AAL65711 regionimmunoglobulin light chain kappa variable PEDF, (SEQ ID NO: 22) AAL65710region immunoglobulin light chain kappa variable LEDG, (SEQ ID NO: 31)AAL65709 region PEDF, (SEQ ID NO: 22) immunoglobulin light chain kappavariable LEDG, (SEQ ID NO: 31) AAL65708 region PEDF, (SEQ ID NO: 22)immunoglobulin light chain kappa variable PEDF, (SEQ ID NO: 22) AAL65707region immunoglobulin light chain kappa variable PEDF, (SEQ ID NO: 22)AAL65706 region immunoglobulin light chain kappa variable PEDF, (SEQ IDNO: 22) AAL65705 region immunoglobulin light chain kappa variable PEDF,(SEQ ID NO: 22) AAL65704 region immunoglobulin light chain kappavariable PEDF, (SEQ ID NO: 22) AAL65703 region immunoglobulin kappalight chain variable SEDF, (SEQ ID NO: 24) AAC64146 regionimmunoglobulin kappa light chain variable SEDF, (SEQ ID NO: 24) AAC64144region immunoglobulin kappa light chain variable PEDF, (SEQ ID NO: 22)ABI64139 region anti-pneumococcal capsular polysaccharide AEDV, (SEQ IDNO: 23) AAL04535 immunoglobulin kappa light chain immunoglobulin lightchain kappa variable AEDV, (SEQ ID NO: 23) AAL65722 regionimmunoglobulin light chain kappa variable AEDV, (SEQ ID NO: 23) AAL65720region immunoglobulin light chain V-J region PEDF, (SEQ ID NO: 22)BAA19563 immunoglobulin light chain V-J region AEDE, (SEQ ID NO: 19)BAA19562 immunoglobulin light chain V-J region AEDE, (SEQ ID NO: 19)BAA19561 immunoglobulin light chain V-J region PEDF, (SEQ ID NO: 22)BAA19560 immunoglobulin light chain V-J region PEDF, (SEQ ID NO: 22)BAA19559 immunoglobulin light chain V-J region AEDV, (SEQ ID NO: 23)BAA19558 immunoglobulin light chain V-J region PEDI, (SEQ ID NO: 21)BAA19556 immunoglobulin kappa light chain variable PEDF, (SEQ ID NO: 22)AAA71907 region immunoglobulin kappa light chain variable AEDV, (SEQ IDNO: 23) AAA71905 region immunoglobulin G1 Fab light chain variable AEDV,(SEQ ID NO: 23) BAF49281 region immunoglobulin G1 Fab light chainvariable PEDF, (SEQ ID NO: 22) BAF48998 region immunoglobulin G1 Fablight chain variable PEDF, (SEQ ID NO: 22) BAF48996 region kappa lightchain V-region AEDM, (SEQ ID NO: 32) CAA37675 immunogloburin G1 Fablight chain variable SEDF, (SEQ ID NO: 24) BAF48994 regionimmunogloburin G1 Fab light chain variable PEDF, (SEQ ID NO: 22)BAF48992 region Ig kappa chain precursor V-J-C region AEDV, (SEQ ID NO:23) A53261 Ig kappa chain precursor V region AEDV, (SEQ ID NO: 23)A49137 Ig kappa chain precursor V-I region SEDI, (SEQ ID NO: 29) PN0445Ig kappa chain precursor V-III region (EVI- PEDF, (SEQ ID NO: 22) A3227415) Ig kappa chain V-IV region (Dep) AEDV, (SEQ ID NO: 23) A34153 Igkappa chain V-IV region (Fue) AEDV, (SEQ ID NO: 23) B34153 Ig kappachain V-II region (Pec) AEDV, (SEQ ID NO: 23) C34153 Chain L, Igg FabFragment (Cd25-Binding). AEDA, (SEQ ID NO: 62) 1MIM_L Chain H, Igg FabFragment (Cd25-Binding). HEDS, (SEQ ID NO: 33) 1MIM_H Ig mu chain Cregion, secreted splice form CEDD, (SEQ ID NO: 34) MHHU immunoglobulinkappa-chain VJ region AEDV, (SEQ ID NO: 23) AAA58923 recombinantmonoclonal antibody IgM 12 PEDF, (SEQ ID NO: 22) ABA41551 kappa lightchain variable region immunoglobulin light chain AEDE, (SEQ ID NO: 19)CAA65054 immunoglobulin light chain lambda variable AEDE, (SEQ ID NO:19) AAL65769 region immunoglobulin light chain lambda variable AEDE,(SEQ ID NO: 19) AAL65767 region immunoglobulin light chain lambdavariable AEDE, (SEQ ID NO: 19) AAL65765 region immunoglobulin lightchain lambda variable TEDE, (SEQ ID NO: 16) AAL65764 regionimmunoglobulin light chain lambda variable AEDE, (SEQ ID NO: 19)AAL65763 region immunoglobulin light chain lambda variable SEDE, (SEQ IDNO: 18) AAL65762 region immunoglobulin light chain lambda variable SEDE,(SEQ ID NO: 18) AAL65761 region immunoglobulin light chain lambdavariable SEDE, (SEQ ID NO: 18) AAL65760 region immunoglobulin lightchain lambda variable AEDE, (SEQ ID NO: 19) AAL65759 regionimmunoglobulin light chain lambda variable AEDE, (SEQ ID NO: 19)AAL65758 region immunoglobulin light chain V-J region PEDF, (SEQ ID NO:22) BAA19563 immunoglobulin light chain V-J region AEDE, (SEQ ID NO: 19)BAA19562 immunoglobulin light chain V-J region AEDE, (SEQ ID NO: 19)BAA19561 immunoglobulin light chain V-J region PEDF, (SEQ ID NO: 22)BAA19560 immunoglobulin light chain V-J region PEDF, (SEQ ID NO: 22)BAA19559 immunoglobulin light chain V-J region AEDV, (SEQ ID NO: 23)BAA19558 immunoglobulin light chain V-J region PEDI, (SEQ ID NO: 21)BAA19556 30-lambda immunoglobulin light chain AEDE, (SEQ ID NO: 19)AAK95335 variable region PREDICTED: similar to Low affinity QEDS, (SEQID NO: 35) XP_001129584 immunoglobulin gamma Fc region receptor II-aprecursor (Fc-gamma RII-a) (FcRII-a) (IgG Fc receptor II-a)(Fc-gamma-RIIa) (CD32 antigen) (CDw32) Fc fragment of IgG, high affinityIa, receptor REDS, (SEQ ID NO: 36) NP_000557 (CD64) TEDG, (SEQ ID NO:37) QEDR, (SEQ ID NO: 38) Fc fragment of IgG, low affinity IIb, receptorQEDS, (SEQ ID NO: 35) NP_001002273 for (CD32) isoform 2 XP_943944 Fcfragment of IgG, low affinity IIb, receptor QEDS, (SEQ ID NO: 35)NP_003992 for (CD32) isoform 1 Fc fragment of IgG, low affinity IIb,receptor QEDS, (SEQ ID NO: 35) NP_001002275 for (CD32) isoform 4 Fcfragment of IgG, low affinity IIb, receptor QEDS, (SEQ ID NO: 35)NP_001002274 for (CD32) isoform 3 XP_001129592 Fc fragment of IgG, highaffinity Ib, receptor QEDR, (SEQ ID NO: 38) NP_001017986 (CD64) isoforma Fc fragment of IgG, high affinity Ib, receptor QEDR, (SEQ ID NO: 38)NP_001004340 (CD64) isoform b XP_496386 Fc fragment of IgG, low affinityIIa, receptor QEDS, (SEQ ID NO: 35) NP_067674 (CD32) XP_943942 lowaffinity immunoglobulin gamma Fc TEDL, (SEQ ID NO: 39) NP_000561 regionreceptor III-B precursor PEDN, (SEQ ID NO: 40) EEDP, (SEQ ID NO: 41) Fcfragment of IgG, low affinity IIIa, TEDL, (SEQ ID NO: 39) NP_000560receptor for (CD16) PEDN, (SEQ ID NO: 40) XP_001133750 EEDP, (SEQ ID NO:41) Low affinity immunoglobulin gamma Fc QEDS, (SEQ ID NO: 35) P12318region receptor II-a precursor (Fc-gamma RII-a) (FcRII-a) (IgG Fcreceptor II-a) (Fc- gamma-RIIa) (CD32 antigen) (CDw32) Low affinityimmunoglobulin gamma Fc TEDL, (SEQ ID NO: 39) O75015 region receptorIII-B precursor (IgG Fc PEDN, (SEQ ID NO: 40) receptor III-1) (Fc-gammaRIII-beta) (Fc- EEDP, (SEQ ID NO: 41) gamma RIIIb) (FcRIIIb) (Fc-gammaRIII) (FcRIII) (FcR-10) (CD16b antigen) Low affinity immunoglobulingamma Fc TEDL, (SEQ ID NO: 39) P08637 region receptor III-A precursor(IgG Fc PEDN, (SEQ ID NO: 40) receptor III-2) (Fc-gamma RIII-alpha) (Fc-EEDP, (SEQ ID NO: 41) gamma RIIIa) (FcRIIIa) (Fc-gamma RIII) (FcRIII)(FcR-10) (CD16a antigen) High affinity immunoglobulin gamma Fc REDS,(SEQ ID NO: 36) P12314 receptor I precursor (Fc-gamma RI) (FcRI) TEDG,(SEQ ID NO: 37) (IgG Fc receptor I) (CD64 antigen). QEDR, (SEQ ID NO:38) IGHG1 immunoglobulin heavy constant AEDT, (SEQ ID NO: 14) Q6PJA4gamma 1 (G1m marker) apoAI [Homo sapiens] LEDL, (SEQ ID NO: 42) CAA01253apolipoprotein C-III precursor [Homo AEDA, (SEQ ID NO: 62) NP_000031sapiens] apolipoprotein A-IV precursor [Homo AEDV, (SEQ ID NO: 23)NP_000473 sapiens]. gelsolin (amyloidosis, Finnish type) [Homo TEDT,(SEQ ID NO: 30) CAM20459 sapiens] KEDA, (SEQ ID NO: 43) SEDC, (SEQ IDNO: 44) QEDL, (SEQ ID NO: 63) gelsolin (amyloidosis, Finnish type) [HomoTEDT, (SEQ ID NO: 30) CAI14413 sapiens] KEDA, (SEQ ID NO: 43) SEDC, (SEQID NO: 44) QEDL, (SEQ ID NO: 63) gelsolin (amyloidosis, Finnish type),isoform TEDT, (SEQ ID NO: 30) EAW87491 CRA_c [Homo sapiens]. KEDA, (SEQID NO: 43) SEDC, (SEQ ID NO: 44) QEDL, (SEQ ID NO: 63) gelsolin(amyloidosis, Finnish type), isoform TEDT, (SEQ ID NO: 30) EAW87490CRA_b [Homo sapiens] KEDA, (SEQ ID NO: 43) SEDC, (SEQ ID NO: 44) QEDL,(SEQ ID NO: 63) gelsolin (amyloidosis, Finnish type), isoform TEDT, (SEQID NO: 30) EAW87489 CRA_a [Homo sapiens] KEDA, (SEQ ID NO: 43) SEDC,(SEQ ID NO: 44) QEDL, (SEQ ID NO: 63) amyloid precursor protein; APP[Homo AEDV, (SEQ ID NO: 23) AAB23646 sapiens]. amyloid precursorprotein; APP [Homo AEDV, (SEQ ID NO: 23) AAB19991 sapiens]. amyloidpeptide AEDV, (SEQ ID NO: 23) AAA51768 Amyloid beta A4 protein precursor(APP) EEDD, (SEQ ID NO: 45) P05067 (ABPP) (Alzheimer disease amyloidprotein) SEDK, (SEQ ID NO: 46) (Cerebral vascular amyloid peptide)(CVAP) DEDD, (SEQ ID NO: 47) (Protease nexin-II) (PN-II) (APPI) (PreA4)DEDG, (SEQ ID NO: 48) [Contains: Soluble APP-alpha (S-APP- AEDV, (SEQ IDNO: 23) alpha); Soluble APP-beta (S-APP-beta); C99; Beta-amyloid protein42 (Beta-APP42); Beta-amyloid protein 40 (Beta-APP40); C83; P3(42);P3(40); Gamma-CTF(59) (Gamma- secretase C-terminal fragment 59) (Amyloidintracellular domain 59) (AID(59)) (AICD- 59); Gamma-CTF(57)(Gamma-secretase C- terminal fragment 57) (Amyloid intracellular domain57) (AID(57)) (AICD-57); Gamma- CTF(50) (Gamma-secretase C-terminalfragment 50) (Amyloid intracellular domain 50) (AID(50)) (AICD-50);C31]. APP protein [Homo sapiens]. EEDD, (SEQ ID NO: 45) AAH65523 SEDK,(SEQ ID NO: 46) DEDD, (SEQ ID NO: 47) DEDG, (SEQ ID NO: 48) APP protein[Homo sapiens]. EEDD, (SEQ ID NO: 45) AAH04369 SEDK, (SEQ ID NO: 46)DEDD, (SEQ ID NO: 47) DEDG, (SEQ ID NO: 48) amyloid beta (A4) precursorprotein EEDD, (SEQ ID NO: 45) AAW82435 (protease nexin-II, Alzheimerdisease) SEDK, (SEQ ID NO: 46) [Homo sapiens]. DEDD, (SEQ ID NO: 47)DEDG, (SEQ ID NO: 48) AEDV, (SEQ ID NO: 23) Calcitonin SEDE, (SEQ ID NO:18) AAA58403 calcitonin precursor SEDE, (SEQ ID NO: 18) AAA35501preprocalcitonin [Homo sapiens] SEDE, (SEQ ID NO: 18) CAA25103Preprocalcitonin SEDE, (SEQ ID NO: 18) AAA51913 Calcitonin precursor[Contains: Calcitonin; SEDE, (SEQ ID NO: 18) P01258 Katacalcin(Calcitonin carboxyl-terminal peptide) (CCP) (PDN-21)] calcitoninisoform CALCA preproprotein SEDE, (SEQ ID NO: 18) NP_001029124 [Homosapiens]. calcitonin isoform CALCA preproprotein SEDE, (SEQ ID NO: 18)NP_001732 [Homo sapiens]. calcitonin isoform CGRP preproprotein SEDE,(SEQ ID NO: 18) NP_001029125 [Homo sapiens]. Calcitonin gene-relatedpeptide 1 precursor SEDE, (SEQ ID NO: 18) P06881 (Calcitoningene-related peptide I) (CGRP-I) (Alpha-type CGRP). atrial natriureticfactor LEDE, (SEQ ID NO: 49) AAA35528 atrial natriuretic factorpropeptide [Homo LEDE, (SEQ ID NO: 49) CAA25700 sapiens]. atrialnatriuretic factor LEDE, (SEQ ID NO: 49) 1101403A Atrial natriureticfactor precursor (ANF) LEDE, (SEQ ID NO: 49) P01160 (Atrial natriureticpeptide) (ANP) (Prepronatriodilatin) (CDD-ANF) [Contains:Cardiodilatin-related peptide (CDP)]. atrial natriuretic peptide LEDE,(SEQ ID NO: 49) AAA35529 keratin [Homo sapiens] GEDA, (SEQ ID NO: 50)AAB30058 keratin [Homo sapiens]. VEDF, (SEQ ID NO: 51) CAA31695 YEDE,(SEQ ID NO: 52) Keratin IEDL, (SEQ ID NO: 53) AAB59562 GEDA, (SEQ ID NO:50) Keratin, type II cytoskeletal 6C (Cytokeratin- VEDL, (SEQ ID NO: 64)P48668 6C) (CK 6C) (K6c keratin) (Cytokeratin-6E) YEDE, (SEQ ID NO: 52)(CK 6E) (Keratin K6h). LEDA, (SEQ ID NO: 65) fibrinogen [Homo sapiens]WEDY, (SEQ ID NO: 54) CAA50740 fibrinogen alpha subunit precursor [HomoDEDW, (SEQ ID NO: 55) AAC97142 sapiens]. SEDL, (SEQ ID NO: 56) YEDQ,(SEQ ID NO: 57) SEDG, (SEQ ID NO: 66) LEDW, (SEQ ID NO: 58) Fibrinogenalpha chain [Homo sapiens] DEDW, (SEQ ID NO: 55) AAI01936 SEDL, (SEQ IDNO: 56) YEDQ, (SEQ ID NO: 57) SEDG, (SEQ ID NO: 66) Fibrinogen alphachain [Homo sapiens] DEDW, (SEQ ID NO: 55) AAH98280 SEDL, (SEQ ID NO:56) YEDQ, (SEQ ID NO: 57) SEDG, (SEQ ID NO: 66) fibrinogen alpha chain,isoform CRA_b DEDW, (SEQ ID NO: 55) EAX04926 [Homo sapiens]. SEDL, (SEQID NO: 56) YEDQ, (SEQ ID NO: 57) SEDG, (SEQ ID NO: 66) LEDW, (SEQ ID NO:58) fibrinogen alpha chain, isoform CRA_c DEDW, (SEQ ID NO: 55) EAX04928[Homo sapiens]. SEDL, (SEQ ID NO: 56) YEDQ, (SEQ ID NO: 57) SEDG, (SEQID NO: 66) fibrinogen alpha chain, isoform CRA_a DEDW, (SEQ ID NO: 55)EAX04924 [Homo sapiens] SEDL, (SEQ ID NO: 56) prion protein precursor;PRNP [Homo YEDR, (SEQ ID NO: 59) AAC62750 sapiens] Major prion proteinprecursor (PrP) (PrP27- YEDR, (SEQ ID NO: 59) P04156 30) (PrP33-35C)(ASCR) (CD230 antigen) prion protein preproprotein [Homo sapiens]. YEDR,(SEQ ID NO: 59) NP_000302 prolactin [Homo sapiens] PEDK, (SEQ ID NO: 60)CAA38264 Prolactin [Homo sapiens]. PEDK, (SEQ ID NO: 60) AAH88370VI. Amyloid Peptides for Active Immunization

Therapeutic agents for use in the methods of the invention areimmunogenic peptides, such as AA peptides and AL peptides, that onadministration to a patient generate antibodies that specifically bindto one or more epitopes comprising X₁EDX₂, such as, for example,epitopes between residues 70-76 of AA (“AA agents”). Additional examplesof agents include immunogenic peptides that comprise a fragmentconsisting of X₁EDX₂ derived from other amyloid proteins (“X₁EDX₂fragments”), such as AL Vκ fragments consisting of the amino acidsequence PEDI, (SEQ ID NO: 21), PEDF, (SEQ ID NO: 22), AEDV, (SEQ ID NO:23), SEDF, (SEQ ID NO: 24), or SEDA, (SEQ ID NO: 25), and AL Vλfragments consisting of the amino acid sequence SEDE, (SEQ ID NO: 18),AEDE, (SEQ ID NO: 19), TEDE, (SEQ ID NO: 16) or PEDE, (SEQ ID NO: 20).An AL Vλ fragment consisting of the amino acid sequence FEDD, (SEQ IDNO: 17) may also be used. Some suitable amyloid proteins include Serumamyloid A protein, immunoglobulin light chain protein, human isletamyloid precursor polypeptide (IAPP), beta amyloid peptide,transthyretin (TTR), ApoA1 and other amyloid proteins listed in Table 1and which comprise the sequence X₁EDX₂. In some agents X₁ is H, T, F, S,P, A or any other amino acid residue immediately preceding ED in anamyloid protein; and X₂ is T, S, E, R, I, V, F, D, A or any other aminoacid residue immediately following ED in such amyloid protein. In someagents, X₁ is H, T, F, S, P, or A and X₂ is T, S, E, D, R, I, V, F or A.In some such agents, when X₁ is H, X₂ is T or A; when X₁ is A, X₂ is S,T, E or V; when X₁ is T, X₂ is E; when X₁ is F, X₂ is D; when X₁ is S,X₂ is E, F or A; and when X₁ is P, X₂ is E, I or F. In some agents, X₁is H, T, F, S, P, or A and X₂ is T, S, E, D, R, I, V, F or A, with theproviso that if X₁ is A, X₂ is not V. In some agents, when X₁ is A, X₂is S, T or E.

Some agents comprise the amino acid sequence GHEDT, (SEQ ID NO: 3),HEDT, (SEQ ID NO: 12), AEDS, (SEQ ID NO: 13), AEDT, (SEQ ID NO: 14),HEDA, (SEQ ID NO: 15), TEDE, (SEQ ID NO: 16), FEDD, (SEQ ID NO: 17),SEDE, (SEQ ID NO: 18), AEDE, (SEQ ID NO: 19), PEDE, (SEQ ID NO: 20),PEDI, (SEQ ID NO: 21), PEDF, (SEQ ID NO: 22), AEDV, (SEQ ID NO: 23),SEDF, (SEQ ID NO: 24) or SEDA, (SEQ ID NO: 25). Some agents consist ofan amino acid sequence selected from the group consisting of GHEDT, (SEQID NO: 3, HEDT, (SEQ ID NO: 12), AEDS, (SEQ ID NO: 13), AEDT, (SEQ IDNO: 14), HEDA, (SEQ ID NO: 15), TEDE, (SEQ ID NO: 16), FEDD, (SEQ ID NO:17), SEDE, (SEQ ID NO: 18), AEDE, (SEQ ID NO: 19), PEDE, (SEQ ID NO:20), PEDI, (SEQ ID NO: 21), PEDF, (SEQ ID NO: 22), AEDV, (SEQ ID NO:23), SEDF, (SEQ ID NO: 24), or SEDA, (SEQ ID NO: 25), linked to acarrier to form a conjugate. Some agents comprise the amino acidsequence GHEDT, (SEQ ID NO: 3, HEDT, (SEQ ID NO: 12), AEDS, (SEQ ID NO:13), AEDT, (SEQ ID NO: 14), HEDA, (SEQ ID NO: 15), TEDE, (SEQ ID NO:16), FEDD, (SEQ ID NO: 17), SEDE, (SEQ ID NO: 18), AEDE, (SEQ ID NO:19), PEDE, (SEQ ID NO: 20), PEDI, (SEQ ID NO: 21), PEDF, (SEQ ID NO:22), AEDV, (SEQ ID NO: 23), SEDF, (SEQ ID NO: 24), or SEDA, (SEQ ID NO:25). Some agents consist of an amino acid sequence selected from thegroup consisting of GHEDT, (SEQ ID NO: 3, HEDT, (SEQ ID NO: 12), AEDS,(SEQ ID NO: 13), AEDT, (SEQ ID NO: 14), HEDA, (SEQ ID NO: 15), TEDE,(SEQ ID NO: 16), FEDD, (SEQ ID NO: 17), SEDE, (SEQ ID NO: 18), AEDE,(SEQ ID NO: 19), PEDE, (SEQ ID NO: 20), PEDI, (SEQ ID NO: 21), PEDF,(SEQ ID NO: 22), SEDF, (SEQ ID NO: 24) and SEDA, (SEQ ID NO: 25), linkedto a carrier to form a conjugate. Some agents comprise an amino acidsequence selected from the group consisting of GHEDT, (SEQ ID NO: 3,HEDT, (SEQ ID NO: 12), AEDS, (SEQ ID NO: 13), AEDT, (SEQ ID NO: 14),HEDA, (SEQ ID NO: 15) and TEDE, (SEQ ID NO: 16).

Preferred AA fragments are human AA1 (HAA1) alpha isoform residues 70-76(GHGAEDS, SEQ ID NO:4), HAA1 beta isoform residues 70-76 (GHDAEDS, SEQID NO:5), HAA1 gamma isoform residues 70-76 (GHDAEDS, SEQ ID NO: 5),HAA2 alpha and beta isoforms residues 70-76 (GHGAEDS, SEQ ID NO: 4),HAA3 residues 70-76 (GDHAEDS, SEQ ID NO:7), HAA4 residues 78-84(STVIEDS, SEQ ID NO:8), mouse AA1 (MAA1) residues 69-75 (GRGHEDT, SEQ IDNO:9), MAA2 residues 69-75 (GRGHEDT, SEQ ID NO: 9), MAA3 residues 62-68(GHGAEDS, SEQ ID NO:10), and MAA4 residues 76-82 (NHGLETL, SEQ ID NO:11)or subfragments of at least three contiguous amino acids of any ofthese. Some AA fragments contain no residues of an AA amyloidosispeptide other than the segment designated above. Other AA fragmentscontain additional flanking residues from an AA amyloidosis peptide butcontain no more than 20 or preferably no more than 10 contiguousresidues in total from an AA amyloidosis peptide. Additional preferredX₁EDX₂ and AL fragments include GHEDT, (SEQ ID NO: 3), HEDT, (SEQ ID NO:12), AEDS, (SEQ ID NO: 13), AEDT, (SEQ ID NO: 14), HEDA, (SEQ ID NO:15), and TEDE, (SEQ ID NO: 16).

Therapeutic agents for use in the methods of the invention also includeimmunogenic AA peptides that on administration to a patient generateantibodies that specifically bind to N-terminal epitopes of AA.Preferred agents induce an immunogenic response directed to an epitopewithin residues 1-15 of human AA.

Preferably, the fragment of AA or AL or other agents such as X₁EDX₂fragments administered lack an epitope that would generate a T-cellresponse to the fragment. Generally, T-cell epitopes are greater than 10contiguous amino acids. Therefore, preferred fragments of amyloidproteins such as AA or X₁EDX₂ fragments are of size 4-10 or preferably7-10 contiguous amino acids; i.e., sufficient length to generate anantibody response without generating a T-cell response. Absence ofT-cell epitopes is preferred because these epitopes are not needed forimmunogenic activity of fragments, and may cause an undesiredinflammatory response in a subset of patients (Anderson et al., (2002)J. Immunol. 168, 3697-3701; Senior (2002) Lancet Neurol. 1, 3).

Preferred AA fragments are human AA1 (HAA1) alpha isoform residues 70-76(GHGAEDS) (SEQ ID NO: 4), HAA1 beta isoform residues 70-76 (GHDAEDS)(SEQ ID NO:5), HAA1 gamma isoform residues 70-76 (GHDAEDS, SEQ ID NO:5), HAA2 alpha and beta isoforms residues 70-76 (GHGAEDS, SEQ ID NO: 4),HAA3 residues 70-76 (GDHAEDS) (SEQ ID NO:7), HAA4 residues 78-84(STVIEDS) (SEQ ID NO:8), mouse AA1 (MAA1) residues 69-75 (GRGHEDT) (SEQID NO:9), MAA2 residues 69-75 (GRGHEDT, SEQ ID NO: 9), MAA3 residues62-68 (GHGAEDS) (SEQ ID NO:10), and MAA4 residues 76-82 (NHGLETL) (SEQID NO:11) or subfragments of at least three contiguous amino acids ofany of these. Some AA fragments contain no residues of an AA amyloidosispeptide other than the segment designated above. Other AA fragmentscontain additional flanking residues from an AA amyloidosis peptide butcontain no more than 20 or preferably no more than 10 contiguousresidues in total from an AA amyloidosis peptide. Additional preferredX₁EDX₂ and AL fragments include GHEDT, (SEQ ID NO: 3), HEDT, (SEQ ID NO:12), AEDS, (SEQ ID NO: 13), AEDT, (SEQ ID NO: 14), HEDA, (SEQ ID NO:15), and TEDE, (SEQ ID NO: 16).

Analogs of the natural AA amyloidosis, AL amyloidosis, and otheramyloidosis peptides can also be used to induce an immune response inthe methods and compositions of the invention. Analogs includingallelic, species and induced variants. Analogs of AA induce antibodiesthat specifically bind with a natural AA 70-76 peptide. Some suchanalogs fail to induce antibodies that specifically binds to epitopesoutside AA70-76. Analogs of AA typically differ from naturally occurringpeptides at up to 30% of amino acid positions by up to 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 position changes. Each deletion or substitution of anatural amino acid residue is considered a position change as is theinsertion of a residue without substitution. Amino acids substitutionsare often conservative substitutions.

Some analogs of AA or AA fragments or AL or AL fragments or otheramyloid protein fragments such as X₁EDX₂ fragments also includeunnatural amino acids or modifications of N or C terminal amino acids atone, two, five, ten or even all positions. For example, the naturalaspartic acid residue can be replaced with iso-aspartic acid. Examplesof unnatural amino acids are D, alpha, alpha-disubstituted amino acids,N-alkyl amino acids, lactic acid, 4-hydroxyproline,gamma-carboxyglutamate, epsilon-N,N,N-trimethyllysine,epsilon-N-acetyllysine, O-phosphoserine, N-acetylserine,N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,omega-N-methylarginine, β-alanine, ornithine, norleucine, norvaline,hydroxyproline, thyroxine, gamma-amino butyric acid, homoserine,citrulline, and isoaspartic acid. Some therapeutic agents of theinvention are all-D peptides, e.g., all-D AA or all-D AA fragments, andall-D peptide analogs. Some therapeutic agents of the invention are 90%all-D peptides, e.g., 90% all-D AA or 90% all-D AA fragments, and 90%all-D peptide analogs. Some therapeutic agents of the invention are 80%all-D peptides, e.g., 80% all-D AA or 80% all-D AA fragments, and 80%all-D peptide analogs. Fragments and analogs can be screened forprophylactic or therapeutic efficacy in transgenic animal models incomparison with untreated or placebo controls as described below.

AA, AL, their fragments, and analogs and X₁EDX₂ fragments and theiranalogs can be synthesized by solid phase peptide synthesis orrecombinant expression, or can be obtained from natural sources.Automatic peptide synthesizers are commercially available from numeroussuppliers, such as Applied Biosystems, Foster City, Calif. Recombinantexpression can be in bacteria, such as E. coli, yeast, insect cells ormammalian cells. Procedures for recombinant expression are described bySambrook et al., Molecular Cloning: A Laboratory Manual (C.S.H.P. Press,NY 2d ed., 1989.)

Therapeutic agents also include longer polypeptides that include, forexample, an immunogenic fragment of AA peptide, AL peptide or an X₁EDX₂fragment, together with one or more other amino acids flanking the AApeptide, AL peptide or X₁EDX₂ fragment on one or one or both sides. Forexample, preferred agents include fusion proteins comprising a segmentof AA, AL or X₁EDX₂ fragment fused to a heterologous amino acid sequencethat induces a helper T-cell response against the heterologous aminoacid sequence and thereby a B-cell response against the AA segment, ALsegment or X₁EDX₂ fragment. One or more flanking heterologous aminoacids can also be used to cap an AA or AL peptide or X₁EDX₂ fragment toprotect it from degradation in manufacture, storage or use. Suchpolypeptides can be screened for prophylactic or therapeutic efficacy inanimal models in comparison with untreated or placebo controls asdescribed below. Therapeutic agents of the invention include animmunogenic fragment of AA or AL or X₁EDX₂ fragment flanked bypolylysine sequences. The polylysine sequences can be fused to theN-terminus, the C terminus, or both the N- and C-terminus of AA or AL oran immunogenic fragment of AA or AL or X₁EDX₂ fragment. The AA or ALpeptide, X₁EDX₂ fragment, analog, active fragment of AA or otherpolypeptide can be administered in associated or multimeric form or indissociated form. Therapeutic agents also include multimers of monomericimmunogenic agents.

In a further variation, an immunogenic fragment of AA or AL or X₁EDX₂fragment can be presented by a virus or a bacterium as part of animmunogenic composition. A nucleic acid encoding the immunogenic peptideis incorporated into a genome or episome of the virus or bacteria.Optionally, the nucleic acid is incorporated in such a manner that theimmunogenic peptide is expressed as a secreted protein or as a fusionprotein with an outer surface protein of a virus or a transmembraneprotein of a bacterium so that the peptide is displayed. Viruses orbacteria used in such methods should be nonpathogenic or attenuated.Suitable viruses include adenovirus, HSV, Venezuelan equine encephalitisvirus and other alpha viruses, vesicular stomatitis virus, and otherrhabdo viruses, vaccinia and fowl pox. Suitable bacteria includeSalmonella and Shigella. Fusion of an immunogenic peptide to HBsAg ofHBV is particularly suitable.

Therapeutic agents also include peptides and other compounds that do notnecessarily have a significant amino acid sequence similarity with AA orAL or X₁EDX₂ fragment but nevertheless serve as mimetics of AA or AL orX₁EDX₂ fragment and induce a similar immune response. For example, anypeptides and proteins forming β-pleated sheets can be screened forsuitability. Anti-idiotypic antibodies against monoclonal antibodies toAA or AL or other amyloidogenic peptides such as or X₁EDX₂ fragments canalso be used. Such anti-Id antibodies mimic the antigen and generate animmune response to it (see Essential Immunology (Roit ed., BlackwellScientific Publications, Palo Alto, 6th ed.), p. 181). Agents other thanAA peptides should induce an immunogenic response against one or more ofthe preferred segments of AA listed above (e.g., AA70-76 or GHEDT, (SEQID NO: 3) or an AL or X₁EDX₂ fragment listed above, such as, forexample, HEDT, (SEQ ID NO: 12), AEDS, (SEQ ID NO: 13), AEDT, (SEQ ID NO:14), HEDA, (SEQ ID NO: 15) and TEDE, (SEQ ID NO: 16).

Preferably, such agents induce an immunogenic response that isspecifically directed to one of these segments without being directed toother segments of AA or AL or amyloid protein from which the X₁EDX₂fragment was derived.

Random libraries of peptides or other compounds can also be screened forsuitability. Combinatorial libraries can be produced for many types ofcompounds that can be synthesized in a step-by-step fashion. Suchcompounds include polypeptides, beta-turn mimetics, polysaccharides,phospholipids, hormones, prostaglandins, steroids, aromatic compounds,heterocyclic compounds, benzodiazepines, oligomeric N-substitutedglycines and oligocarbamates. Large combinatorial libraries of thecompounds can be constructed by the encoded synthetic libraries (ESL)method described in Affymax, WO 95/12608, Affymax, WO 93/06121, ColumbiaUniversity, WO 94/08051, Pharmacopeia, WO 95/35503 and Scripps, WO95/30642 (each of which is incorporated by reference for all purposes).Peptide libraries can also be generated by phage display methods. See,e.g., Devlin, WO 91/18980.

Combinatorial libraries and other compounds are initially screened forsuitability by determining their capacity to specifically bind toantibodies or lymphocytes (B or T) known to be specific for AA or otheramyloidogenic peptides. For example, initial screens can be performedwith any polyclonal sera or monoclonal antibody to AA or AL or afragment thereof or to an X₁EDX₂ fragment. Compounds can then bescreened for specifically binding to a specific epitope within AA (e.g.,AA70-76 or GHEDT, (SEQ ID NO: 3) or AL or to an X₁EDX₂ fragment listedabove, such as, for example, HEDT, (SEQ ID NO: 12), AEDS, (SEQ ID NO:13), AEDT, (SEQ ID NO: 14), HEDA, (SEQ ID NO: 15) and TEDE, (SEQ ID NO:16).

Compounds can be tested by the same procedures described for mappingantibody epitope specificities. Compounds identified by such screens arethen further analyzed for capacity to induce antibodies or reactivelymphocytes to AA or AL or fragments thereof or to an X₁EDX₂ fragment.For example, multiple dilutions of sera can be tested on microtiterplates that have been precoated with AA or AL or a fragment thereof oran X₁EDX₂ fragment and a standard ELISA can be performed to test forreactive antibodies to AA or AL or the fragment or to the X₁EDX₂fragment. Compounds can then be tested for prophylactic and therapeuticefficacy in transgenic animals predisposed to amyloidosis, such as, forexample, AA Amyloidosis or AL amyloidosis. The same screening approachcan be used on other potential agents, analogs of AA, analogs of AL andlonger peptides, including fragments of AA, AL and X₁EDX₂ fragments,described above.

VII. Conjugates

Some agents for inducing an immune response contain the appropriateepitope for inducing an immune response against AA but are too small tobe immunogenic. In this situation, a peptide immunogen can be linked toa suitable carrier molecule to form a conjugate which helps elicit animmune response. A single agent can be linked to a single carrier,multiple copies of an agent can be linked to multiple copies of acarrier, which are in turn linked to each other, multiple copies of anagent can be linked to a single copy of a carrier, or a single copy ofan agent can be linked to multiple copies of a carrier, or differentcarriers. Suitable carriers include serum albumins, keyhole limpethemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanustoxoid, or a toxoid from other pathogenic bacteria, such as diphtheria,E. coli, cholera, or H. pylori, or an attenuated toxin derivative. Tcell epitopes are also suitable carrier molecules. Some conjugates canbe formed by linking agents of the invention to an immunostimulatorypolymer molecule (e.g., tripalmitoyl-5-glycerine cysteine (Pam₃Cys),mannan (a manose polymer), or glucan (a beta 1→2 polymer)), cytokines(e.g., IL-1, IL-1 alpha and beta peptides, IL-2, gamma-INF, IL-10,GM-CSF), and chemokines (e.g., MIP1 alpha and beta, and RANTES).Immunogenic agents can also be linked to peptides that enhance transportacross tissues, as described in O'Mahony, WO 97/17613 and WO 97/17614.Immunogens may be linked to the carries with or with out spacers aminoacids (e.g., gly-gly).

Some conjugates can be formed by linking agents of the invention to atleast one T cell epitope. Some T cell epitopes are promiscuous whileother T cell epitopes are universal. Promiscuous T cell epitopes arecapable of enhancing the induction of T cell immunity in a wide varietyof subjects displaying various HLA types. In contrast to promiscuous Tcell epitopes, universal T cell epitopes are capable of enhancing theinduction of T cell immunity in a large percentage, e.g., at least 75%,of subjects displaying various HLA molecules encoded by different HLA-DRalleles.

A large number of naturally occurring T-cell epitopes exist, such as,tetanus toxoid (e.g., the P2 and P30 epitopes), Hepatitis B surfaceantigen, pertussis, toxoid, measles virus F protein, Chlamydiatrachomitis major outer membrane protein, diphtheria toxoid (e.g.,CRM197), Plasmodium falciparum circumsporozite T, Plasmodium falciparumCS antigen, Schistosoma mansoni triose phosphate isomersae, Escherichiacoli TraT, and Influenza virus hemagluttinin (HA). The immunogenicpeptides of the invention can also be conjugated to the T-cell epitopesdescribed in Sinigaglia F. et al., Nature, 336:778-780 (1988); Chicz R.M. et al., J. Exp. Med., 178:27-47 (1993); Hammer J. et al., Cell74:197-203 (1993); Falk K. et al., Immunogenetics, 39:230-242 (1994); WO98/23635; Southwood S. et al. J. Immunology; 160:3363-3373 (1998); and,Giannini, G. et al. Nucleic Acids Res. 12: 4063-4069 (1984), (each ofwhich is incorporated herein by reference for all purposes). Furtherexamples include:

Influenza Hemagluttinin: HA₃₀₇₋₃₁₉ Malaria CS: (SEQ ID NO: 67)T3 epitope EKKIAKMEKASSVFNV,. Hepatitis B surface antigen:(SEQ ID NO: 68) HBsAg₁₉₋₂₈ FFLLTRILTI,. Heat Shock Protein 65:(SEQ ID NO: 69) hsp65₁₅₃₋₁₇₁ DQSIGDLIAEAMDKVGNEG,.  (SEQ ID NO: 70)bacille Calmette-Guerin QVHFQPLPPAVVKL,. Tetanus toxoid: (SEQ ID NO: 71)TT₈₃₀₋₈₄₄ QYIKANSKFIGITEL,. Tetanus toxoid: (SEQ ID NO: 72)TT₉₄₇₋₉₆₇ FNNFTVSFWLRVPKVSASHLE,. HIV gp120 T1: (SEQ ID NO: 73)KQIINMWQEVGKAMYA,. Tetanus toxoid: TT₉₄₇₋₉₆₇ FNNFTVSFWLRVPKVSASHLEHIV gp120 T1: KQIINMWQEVGKAMYA.

Alternatively, the conjugates can be formed by linking agents of theinvention to at least one artificial T-cell epitope capable of binding alarge proportion of MHC Class II molecules., such as the pan DR epitope(“PADRE”). PADRE is described in U.S. Pat. No. 5,736,141, WO 95/07707,and Alexander J et al., Immunity, 1:751-761 (1994) (each of which isincorporated herein by reference for all purposes). A preferred PADREpeptide is AKXVAAWTLKAAA, (SEQ ID NO: 74), (common residues bolded)wherein X is preferably cyclohexylalanine tyrosine or phenylalanine,with cyclohexylalanine being most preferred.

Immunogenic agents can be linked to carriers by chemical crosslinkingTechniques for linking an immunogen to a carrier include the formationof disulfide linkages using N-succinimidyl-3-(2-pyridyl-thio) propionate(SPDP) and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(SMCC) (if the peptide lacks a sulfhydryl group, this can be provided byaddition of a cysteine residue). These reagents create a disulfidelinkage between themselves and peptide cysteine resides on one proteinand an amide linkage through the epsilon-amino on a lysine, or otherfree amino group in other amino acids. A variety of suchdisulfide/amide-forming agents are described by Immun. Rev. 62, 185(1982). Other bifunctional coupling agents form a thioether rather thana disulfide linkage. Many of these thio-ether-forming agents arecommercially available and include reactive esters of 6-maleimidocaproicacid, 2-bromoacetic acid, and 2-iodoacetic acid,4-(N-maleimido-methyl)cyclohexane-1-carboxylic acid. The carboxyl groupscan be activated by combining them with succinimide or1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt.

Immunogenicity can be improved through the addition of spacer residues(e.g., Gly-Gly) between the T_(h) epitope and the peptide immunogen ofthe invention. In addition to physically separating the T_(h) epitopefrom the B cell epitope (i.e., the peptide immunogen), the glycineresidues can disrupt any artificial secondary structures created by thejoining of the T_(h) epitope with the peptide immunogen, and therebyeliminate interference between the T and/or B cell responses. Theconformational separation between the helper epitope and the antibodyeliciting domain thus permits more efficient interactions between thepresented immunogen and the appropriate T_(h) and B cells.

To enhance the induction of T cell immunity in a large percentage ofsubjects displaying various HLA types to an agent of the presentinvention, a mixture of conjugates with different T_(h) cell epitopescan be prepared. The mixture may contain a mixture of at least twoconjugates with different T_(h) cell epitopes, a mixture of at leastthree conjugates with different T_(h) cell epitopes, or a mixture of atleast four conjugates with different T_(h) cell epitopes. The mixturemay be administered with an adjuvant.

Immunogenic peptides can also be expressed as fusion proteins withcarriers (i.e., heterologous peptides). The immunogenic peptide can belinked at its amino terminus, its carboxyl terminus, or both to acarrier. Optionally, multiple repeats of the immunogenic peptide can bepresent in the fusion protein. Optionally, an immunogenic peptide can belinked to multiple copies of a heterologous peptide, for example, atboth the N and C termini of the peptide. Optionally, multiple copies ofan immunogenic peptide can be linked to multiple copies of aheterologous peptide. which are linked to each other. Some carrierpeptides serve to induce a helper T-cell response against the carrierpeptide. The induced helper T-cells in turn induce a B-cell responseagainst the immunogenic peptide linked to the carrier.

Some examples of fusion proteins suitable for use in the invention areshown below. Some of these fusion proteins comprise segments of AAlinked to tetanus toxoid epitopes such as described in U.S. Pat. No.5,196,512, EP 378,881 and EP 427,347. Some fusion proteins comprisesegments of AA linked to at least one PADRE peptide described in U.S.Pat. No. 5,736,142. Some heterologous peptides are promiscuous T-cellepitopes, while other heterologous peptides are universal T-cellepitopes. In some methods, the agent for administration is simply asingle fusion protein with an AA segment linked to a heterologoussegment in linear configuration. The therapeutic agents of the inventioncan be represented using a formula. For example, in some methods, theagent is multimer of fusion proteins represented by the formula 2^(x),in which x is an integer from 1-5. Preferably x is 1, 2 or 3, with 2being most preferred. When x is two, such a multimer has four fusionproteins linked in a preferred configuration referred to as MAP4 (seeU.S. Pat. No. 5,229,490).

The MAP4 configuration is shown below, where branched structures areproduced by initiating peptide synthesis at both the N terminal and sidechain amines of lysine. Depending upon the number of times lysine isincorporated into the sequence and allowed to branch, the resultingstructure will present multiple N termini. In this example, fouridentical N termini have been produced on the branched lysine-containingcore. Such multiplicity greatly enhances the responsiveness of cognate Bcells. In the examples below, Z refers to an immunogenic fragment of AA,AL or an X₁EDX₂ fragment, and Z1-4 refer to immunogenic fragment(s) ofAA, AL or an X₁EDX₂ fragment. The fragments can be the same as eachother or different.

Other examples of fusion proteins include:

-   -   Z-Tetanus toxoid 830-844 in a MAP4 configuration:

(SEQ ID NO: 71) Z-QYIKANSKFIGITEL,

-   -   Z-Tetanus toxoid 947-967 in a MAP4 configuration:

(SEQ ID NO: 72) Z-FNNFTVSFWLRVPKVSASHLE,

-   -   Z-Tetanus toxoid 830-844 in a MAP4 configuration:

(SEQ ID NO: 71) Z-QYIKANSKFIGITEL,

-   -   Z-Tetanus toxoid 830-844+947-967 in a linear configuration:

(SEQ ID NO: 75) Z-QYIKANSKFIGITELFNNFTVSFWLRVPKVSASHLE,.

-   -   PADRE peptide (all in linear configurations), wherein X is        preferably cyclohexylalanine, tyrosine or phenylalanine, with        cyclohexylalanine being most preferred-Z:

(SEQ ID NO: 74) AKXVAAWTLKAAA-Z,.

-   -   Z×3-PADRE peptide:

(SEQ ID NO: 74) Z-Z-Z-AKXVAAWTLKAAA,.

-   -   Z-ovalbumin 323-339 in a linear configuration:

(SEQ ID NO: 76) Z-ISQAVHAAHAEINEAGR,.

Further examples of fusion proteins include:

(SEQ ID NO: 74) AKXVAAWTLKAAA-Z-Z-Z-Z,. (Z-(SEQ ID NO: 74)Z-AKXVAAWTLKAAA,. (SEQ ID NO: 77) PKYVKQNTLKLAT-Z-Z-Z,. (SEQ ID NO: 77)Z-PKYVKQNTLKLAT-Z,. (SEQ ID NO: 77) Z-Z-Z-PKYVKQNTLKLAT,.(Z-Z-(SEQ ID NO: 77) Z-Z-PKYVKQNTLKLAT, (SEQ ID NO: 78)Z-PKYVKQNTLKLAT-EKKIAKMEKASSVFNV-QYIKANSKFIGITEL- FNNFTVSFWLRVPKVSASHLE-(SEQ ID NO: 79) Z-Z-Z-QYIKANSKFIGITEL-FNNFTVSFWLRVPKVSASHLE,.(SEQ ID NO: 79) Z-QYIKANSKFIGITELCFNNFTVSFWLRVPKVSASHLE-Z,.(SEQ ID NO: 79) QYIKANSKFIGITELCFNNFTVSFWLRVPKVSASHLE-Z, (SEQ ID NO: 71)Z-QYIKANSKFIGITEL,on a 2 branched resin: fragments can be the same as each other ordifferent.

The same or similar carrier proteins and methods of linkage can be usedfor generating immunogens to be used in generation of antibodies againstAA or an immunogenic fragment of AA, AL or an X₁EDX₂ fragment. Forexample, AA or an immunogenic fragment of AA, AL or an X₁EDX₂ fragmentlinked to a carrier can be administered to a laboratory animal in theproduction of monoclonal antibodies to AA or an immunogenic fragment ofAA, AL or an X₁EDX₂ fragment.

VIII. Nucleic Acid Encoding Therapeutic Agents

Therapeutic agents of the invention also include nucleic acids. Immuneresponses against amyloid deposits can also be induced by administrationof nucleic acids encoding segments of AA peptide, and fragments thereof,other peptide immunogens such as X₁EDX₂ fragments, or antibodies andtheir component chains, such as antibodies 2A4, 8G9 and 7D8, used forpassive immunization. Such agents for use in the methods of theinvention include nucleic acids encoding AA peptides that onadministration to a patient generate antibodies that specifically bindto one or more epitopes between residues 70-76 of AA, AL or nucleicacids encoding peptides comprising X₁EDX₂ fragments. Such agents for usein the methods of the invention also include nucleic acids encodingantibodies that specially bind to a C-terminal neoepitope of AA or toX₁EDX₂. In particular, such nucleic acids encode antibodies thatspecifically bind to HAA1 alpha isoform within residues 70-76 (GHGAEDS,(SEQ ID NO: 4), HAA1 beta isoform within residues 70-76 (GHDAEDS, (SEQID NO: 5), HAA1 gamma isoform within residues 70-76 (GHDAEDS, (SEQ IDNO: 5), HAA2 alpha and beta isoforms within residues 70-76 (GHGAEDS,(SEQ ID NO: 4), HAA3 within residues 70-76 (GDHAEDS, (SEQ ID NO: 7),HAA4 within residues 78-84 (STVIEDS, (SEQ ID NO: 8), mouse AA1 (MAA1)within residues 69-75 (GRGHEDT, (SEQ ID NO: 9), MAA2 within residues69-75 (GRGHEDT, (SEQ ID NO: 9), MAA3 within residues 62-68 (GHGAEDS,(SEQ ID NO: 4), and MAA4 within residues 76-82 (NHGLETL, (SEQ ID NO:11). Such nucleic acids can be DNA or RNA. Additional preferred nucleicacids encode antibodies that specifically bind to HEDT, (SEQ ID NO: 12),AEDS, (SEQ ID NO: 13), AEDT, (SEQ ID NO: 14), HEDA, (SEQ ID NO: 15) orTEDE, (SEQ ID NO: 16) or other X₁EDX₂ peptides listed above. A nucleicacid segment encoding an immunogen is typically linked to regulatoryelements, such as a promoter and enhancer, that allow expression of theDNA segment in the intended target cells of a patient. For expression inblood cells, as is desirable for induction of an immune response,promoter and enhancer elements from light or heavy chain immunoglobulingenes or the CMV major intermediate early promoter and enhancer aresuitable to direct expression. The linked regulatory elements and codingsequences are often cloned into a vector. For administration ofdouble-chain antibodies, the two chains can be cloned in the same orseparate vectors. The nucleic acids encoding therapeutic agents of theinvention can also encode at least one T cell epitope. The disclosuresherein which relate to the use of adjuvants and the use of carriersapply mutatis mutandis to their use with the nucleic acids encoding thetherapeutic agents of the present invention.

A number of viral vector systems are available including retroviralsystems (see, e.g., Lawrie and Tumin, Cur. Opin. Genet. Develop. 3,102-109 (1993)); adenoviral vectors (see, e.g., Bett et al., J. Virol.67, 5911 (1993)); adeno-associated virus vectors (see, e.g., Zhou etal., J. Exp. Med. 179, 1867 (1994)), viral vectors from the pox familyincluding vaccinia virus and the avian pox viruses, viral vectors fromthe alpha virus genus such as those derived from Sindbis and SemlikiForest Viruses (see, e.g., Dubensky et al., J. Virol. 70, 508-519(1996)), Venezuelan equine encephalitis virus (see U.S. Pat. No.5,643,576) and rhabdoviruses, such as vesicular stomatitis virus (see WO96/34625) and papillomaviruses (Ohe et al., Human Gene Therapy 6,325-333 (1995); Woo et al., WO 94/12629 and Xiao & Brandsma, NucleicAcids. Res. 24, 2630-2622 (1996)).

DNA encoding an immunogen, or a vector containing the same, can bepackaged into liposomes. Suitable lipids and related analogs aredescribed by U.S. Pat. Nos. 5,208,036, 5,264,618, 5,279,833 and5,283,185. Vectors and DNA encoding an immunogen can also be adsorbed toor associated with particulate carriers, examples of which includepolymethyl methacrylate polymers and polylactides andpoly(lactide-co-glycolides), see, e.g., McGee et al., J. Micro Encap.(1996).

Gene therapy vectors or naked DNA can be delivered in vivo byadministration to an individual patient, typically by systemicadministration (e.g., intravenous, intraperitoneal, nasal, gastric,intradermal, intramuscular, subdermal, or intracranial infusion) ortopical application (see e.g., U.S. Pat. No. 5,399,346). Such vectorscan further include facilitating agents such as bupivacine (U.S. Pat.No. 5,593,970). DNA can also be administered using a gene gun. (See Xiao& Brandsma, supra.) The DNA encoding an immunogen is precipitated ontothe surface of microscopic metal beads. The microprojectiles areaccelerated with a shock wave or expanding helium gas, and penetratetissues to a depth of several cell layers. For example, The Accel™ GeneDelivery Device manufactured by Agacetus, Inc. Middleton Wis. issuitable. Alternatively, naked DNA can pass through skin into the bloodstream simply by spotting the DNA onto skin with chemical or mechanicalirritation (see WO 95/05853).

In a further variation, vectors encoding immunogens can be delivered tocells ex vivo, such as cells explanted from an individual patient (e.g.,lymphocytes, bone marrow aspirates, tissue biopsy) or universal donorhematopoietic stem cells, followed by reimplantation of the cells into apatient, usually after selection for cells which have incorporated thevector.

IX. Adjuvants

Immunogenic agents of the invention, such as peptides, are sometimesadministered in combination with an adjuvant. The adjuvant increases thetiter of induced antibodies and/or the binding affinity of inducedantibodies relative to the situation if the peptide were used alone. Avariety of adjuvants can be used in combination with an immunogenicfragment of AA, to elicit an immune response. Preferred adjuvantsaugment the intrinsic response to an immunogen without causingconformational changes in the immunogen that affect the qualitative formof the response. Preferred adjuvants include aluminum hydroxide andaluminum phosphate, 3 De-O-acylated monophosphoryl lipid A (MPL™) (seeGB 2220211 (RIBI ImmunoChem Research Inc., Hamilton, Mont., now part ofCorixa), RC-529 (Corixa, Hamilton, Mont.). STIMULON™ QS-21 is atriterpene glycoside or saponin isolated from the bark of the QuillajaSaponaria Molina tree found in South America (see Kensil et al., inVaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman,Plenum Press, NY, 1995); U.S. Pat. No. 5,057,540), (AquilaBioPharmaceuticals, Framingham, Mass.). Other adjuvants are oil in wateremulsions (such as squalene or peanut oil), optionally in combinationwith immune stimulants, such as monophosphoryl lipid A (see Stoute etal., N. Engl. J. Med. 336, 86-91 (1997)), pluronic polymers, and killedmycobacteria. Another adjuvant is CpG (WO 98/40100). Adjuvants can beadministered as a component of a therapeutic composition with an activeagent or can be administered separately, before, concurrently with, orafter administration of the therapeutic agent.

A preferred class of adjuvants is aluminum salts (alum), such as alumhydroxide, alum phosphate, alum sulfate. Such adjuvants can be used withor without other specific immunostimulating agents such as MPL or 3-DMP,QS-21, polymeric or monomeric amino acids such as polyglutamic acid orpolylysine. Another class of adjuvants is oil-in-water emulsionformulations. Such adjuvants can be used with or without other specificimmunostimulating agents such as muramyl peptides (e.g.,N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE),N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) THERAMIDE™), or other bacterial cell wallcomponents. Oil-in-water emulsions include (a) MF59 (WO 90/14837),containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionallycontaining various amounts of MTP-PE) formulated into submicronparticles using a microfluidizer such as Model 110Y microfluidizer(Microfluidics, Newton Mass.), (b) SAF, containing 10% Squalene, 0.4%Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP, eithermicrofluidized into a submicron emulsion or vortexed to generate alarger particle size emulsion, and (c) RIBI™ adjuvant system (RAS),(Ribi ImmunoChem, Hamilton, Mont.) containing 2% squalene, 0.2% Tween80, and one or more bacterial cell wall components from the groupconsisting of monophosphoryllipid A (MPL), trehalose dimycolate (TDM),and cell wall skeleton (CWS), preferably MPL+CWS (DETOXT™).

Another class of preferred adjuvants is saponin adjuvants, such asSTIMULON™ (QS-21, Aquila, Framingham, Mass.) or particles generatedtherefrom such as ISCOMs (immunostimulating complexes) and ISCOMATRIX.Other adjuvants include RC-529, GM-CSF and Complete Freund's Adjuvant(CFA) and Incomplete Freund's Adjuvant (IFA). Other adjuvants includecytokines, such as interleukins (e.g., IL-1α and β peptides, IL-2, IL-4,IL-6, IL-12, IL13, and IL-15), macrophage colony stimulating factor(M-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF),tumor necrosis factor (TNF), chemokines, such as MIP1α and β and RANTES.Another class of adjuvants is glycolipid analogues includingN-glycosylamides, N-glycosylureas and N-glycosylcarbamates, each ofwhich is substituted in the sugar residue by an amino acid, asimmuno-modulators or adjuvants (see U.S. Pat. No. 4,855,283). Heat shockproteins, e.g., HSP70 and HSP90, may also be used as adjuvants.

An adjuvant can be administered with an immunogen as a singlecomposition, or can be administered before, concurrent with, or afteradministration of the immunogen. Immunogen and adjuvant can be packagedand supplied in the same vial or can be packaged in separate vials andmixed before use. Immunogen and adjuvant are typically packaged with alabel indicating the intended therapeutic application. If immunogen andadjuvant are packaged separately, the packaging typically includesinstructions for mixing before use. The choice of an adjuvant and/orcarrier depends on the stability of the immunogenic formulationcontaining the adjuvant, the route of administration, the dosingschedule, the efficacy of the adjuvant for the species being vaccinated,and, in humans, a pharmaceutically acceptable adjuvant is one that hasbeen approved or is approvable for human administration by pertinentregulatory bodies. For example, Complete Freund's adjuvant is notsuitable for human administration. Alum, MPL and QS-21 are preferred.Optionally, two or more different adjuvants can be used simultaneously.Preferred combinations include alum with MPL, alum with QS-21, MPL withQS-21, MPL or RC-529 with GM-CSF, and alum, QS-21 and MPL together.Also, Incomplete Freund's adjuvant can be used (Chang et al., AdvancedDrug Delivery Reviews 32, 173-186 (1998)), optionally in combinationwith any of alum, QS-21, and MPL and all combinations thereof.

X. Passive Administration of Antibodies

Therapeutic agents of the present invention include antibodies thatspecifically bind to to an epitope comprising X₁EDX₂ in an aggregatedamyloid protein, wherein X₁ is H, T, F, S, P, A or any other amino acidresidue immediately preceding ED in such aggregated amyloid protein; andwherein X₂ is T, S, E, R, I, V, F, A or any other amino acid residueimmediately following ED in such aggregated amyloid protein, includingepitopes within amyloid peptides such as AA. The antibodies used forpassive administration can be antibodies that bind to C-terminal orN-terminal epitopes of AA. Other amyloid proteins in addition to Serumamyloid A protein include serum amyloid A protein, immunoglobulin lightchain protein, such as, for example, Vλ6 Wil or Vκ, human islet amyloidprecursor polypeptide (IAPP), beta amyloid peptide, transthyretin (TTR)and ApoA1, as well as others listed in Table 1 above.

AA is formed by proteolytic cleavage of SAA. Preferred antibodiesspecifically bind to neoepitopes of AA which form upon proteolyticcleavage of SAA. Preferred antibodies specially bind to a C-terminalneoepitope of AA, especially, such antibodies specifically bind to HAA1alpha isoform within residues 70-76 (GHGAEDS, SEQ ID NO:4), HAA1 betaisoform within residues 70-76 (GHDAEDS, SEQ ID NO:5), HAA1 gamma isoformwithin residues 70-76 (GHDAEDS, SEQ ID NO: 5), HAA2 alpha and betaisoforms within residues 70-76 (GHGAEDS, SEQ ID NO: 10), HAA3 withinresidues 70-76 (GDHAEDS, SEQ ID NO:7), HAA4 within residues 78-84(STVIEDS, SEQ ID NO:8), mouse AA1 (MAA1) within residues 69-75 (GRGHEDT,SEQ ID NO:9), MAA2 within residues 69-75 (GRGHEDT, SEQ ID NO: 9), MAA3within residues 62-68 (GHGAEDS, SEQ ID NO:10), and MAA4 within residues76-82 (NHGLETL, SEQ ID NO:11). Some antibodies only bind to an epitopewithin one of these peptides. Other antibodies bind to epitopes withinmore than one of these peptides. For example, some antibodiesspecifically bind to a GHGAEDS, (SEQ ID NO: 4) peptide and a GHDAEDS,SEQ ID NO: 5) peptide. Some antibodies bind to a GHGAEDS, SEQ ID NO: 4)peptide without specifically binding to a GHDAEDS, SEQ ID NO: 5)peptide. Binding to at least one of the human AA peptides is preferable.Binding to at least one of the human AA peptides and a correspondingmouse peptide is useful in that the same antibody can be tested in amouse model and subsequently used in humans. Some preferred antibodiesspecifically bind to epitopes within HAA1 alpha isoform residues 71-76,72-76, 73-76, 74-76, 70-75, 70-74, 70-73, 70-72, 71-75, 72-75, 73-75,71-74, 71-73, 72-74, or MAA1 residues 70-75, 71-75, 72-75, 73-75, 69-74,69-73, 69-72, 69-71, 70-74, 71-74, 72-74, 70-73, 70-72. Such antibodiestypically specifically bind to amyloid deposits but may or may not bindto soluble AA. When an antibody is said to specifically bind to anepitope within specified residues, such as HAA1 alpha isoform residues70-76 of for example, what is meant is that the antibody specificallybinds to a polypeptide containing the specified residues (i.e., residues70-76 of HAA1 alpha isoform in this an example). Such an antibody doesnot necessarily contact every residue within residues 70-76 of HAA1alpha isoform. Nor does every single amino acid substitution or deletionwith in residues 70-76 of HAA1 alpha isoform necessarily significantlyaffect binding affinity. Such neoepitope antibodies bind to AA but notto SAA. Epitope specificity of an antibody can be determined, forexample, as described by WO 00/72880.

The antibodies used for passive administration can be antibodies toN-terminal epitopes of AA. Preferred antibodies specifically bind to aN-terminal neoepitope of AA, especially, such antibodies specificallybind to HAA1 residues 1-15 (RSFFSFLGEAFDGAR, SEQ ID NO. 80), HAA2residues 1-15 (RSFFSFLGEAFDGAR, SEQ ID NO. 80), HAA3 residues 1-15(QGWLTFLKAAGQGAK, SEQ ID NO: 81), HAA4 residues 1-15 (ESWRSFFKEA, (SEQID NO: 82), MAA1 residues 1-15 (GFFSFVHEAFQGAGD, SEQ ID NO: 83), MAA2residues 1-15 (GFFSFVHEAFQGAGD, SEQ ID NO: 83), MAA3 residues 1-9(EAGQGSRD, (SEQ ID NO: 84), and residues 1-14 MAA4 (WYSFFREAVQGTWD, SEQID NO: 85). Some antibodies only bind to an epitope within one of thesepeptides. Other antibodies bind to epitopes within more than one ofthese peptides. For example, some antibodies specifically bind to aRSFFSFLGEAFDGAR, SEQ ID NO: 80) peptide and a QGWLTFLKAAGQGAK, SEQ IDNO: 81) peptide. Some antibodies bind to a RSFFSFLGEAFDGAR, SEQ ID NO:80) peptide without specifically binding to a QGWLTFLKAAGQGAK, SEQ IDNO: 81) peptide. Binding to at least one of the human AA peptides ispreferable. Binding to at least one of the human AA peptides and acorresponding mouse peptide is useful in that the same antibody can betested in a mouse model and subsequently used in humans.

Some antibodies specifically bind to an epitope consisting of suchX₁EDX₂ Preferably such antibodies specifically bind to such epitope inan aggregated amyloid protein. Some of such antibodies preferentiallyspecifically bind to an aggregated amyloid protein relative to themonomeric form of such amyloid protein. In some antibodies, X₁ is H, T,F, S, P, or A and X₂ is T, S, E, D, R, I, V, F or A. In some suchantibodies, when X₁ is H, X₂ is T or A; when X₁ is A, X₂ is S, T, E orV; when X₁ is T, X₂ is E; when X₁ is F, X₂ is D; when X₁ is S, X₂ is E,F or A; and when X₁ is P, X₂ is E, I or F. In some antibodies, X₁ is H,T, F, S, P, or A and X₂ is T, S, E, D, R, I, V, F or A, with the provisothat if X₁ is A, X₂ is not V. In some antibodies, when X₁ is A, X₂ is S,T or E.

Some antibodies specifically bind an epitope comprising the amino acidsequence GHEDT, (SEQ ID NO 3), HEDT, (SEQ ID NO: 12), AEDS, (SEQ ID NO:13), AEDT, (SEQ ID NO: 14), HEDA, (SEQ ID NO: 15), TEDE, (SEQ ID NO:16), FEDD, (SEQ ID NO: 17), SEDE, (SEQ ID NO: 18), AEDE, (SEQ ID NO:19), PEDE, (SEQ ID NO: 20), PEDI, (SEQ ID NO: 21), PEDF, (SEQ ID NO:22), AEDV, (SEQ ID NO: 23), SEDF, (SEQ ID NO: 24) or SEDA, (SEQ ID NO:25).

Some antibodies specifically bind to a peptide comprising an amino acidsequence selected from the group consisting of GHEDT, (SEQ ID NO: 3),HEDT, (SEQ ID NO: 12), AEDS, (SEQ ID NO: 13), AEDT, (SEQ ID NO: 14),HEDA, (SEQ ID NO: 15), TEDE, (SEQ ID NO: 16), FEDD, (SEQ ID NO: 17),SEDE, (SEQ ID NO: 18), AEDE, (SEQ ID NO: 19), PEDE, (SEQ ID NO: 20),PEDI, (SEQ ID NO: 21), PEDF, (SEQ ID NO: 22), SEDF, (SEQ ID NO: 24) andSEDA, (SEQ ID NO: 25). Some antibodies specifically bind to a peptidecomprising an amino acid sequence selected from the group consisting ofGHEDT, (SEQ ID NO: 3), HEDT, (SEQ ID NO: 12), AEDS, (SEQ ID NO: 13),AEDT, (SEQ ID NO: 14), HEDA, (SEQ ID NO: 15) and TEDE, (SEQ ID NO: 16).

Some antibodies are raised to a peptide comprising GHEDT, (SEQ ID NO:3), such as, for example, 2A4, 7D8 and 8G9, or are humanized or chimericversions thereof.

Antibodies can be polyclonal or monoclonal. Polyclonal sera typicallycontain mixed populations of antibodies specifically binding to severalepitopes along the length of AA. However, polyclonal sera can bespecific to a particular segment of AA, such as residues 70-76 of HAA1alpha isoform. Preferred antibodies are chimeric, or humanized (seeQueen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989) and WO90/07861, U.S. Pat. Nos. 5,693,762, 5,693,761, 5,585,089, 5,530,101 andWinter, U.S. Pat. No. 5,225,539), or human (Lonberg et al., WO93/12227(1993); U.S. Pat. Nos. 5,877,397, 5,874,299, 5,814,318, 5,789,650,5,770,429, 5,661,016, 5,633,425, 5,625,126, 5,569,825, 5,545,806, Nature148, 1547-1553 (1994), Nature Biotechnology 14, 826 (1996),Kucherlapati, WO 91/10741 (1991)). An alternative approach forhumanizing an antibody, also known as veneering, is described in U.S.Pat. No. 6,797,492. Several mouse antibodies of different bindingspecificities are available as starting materials for making humanizedantibodies.

Representative humanized antibodies are humanized version 7D8 antibody(ATCC Accession Number PTA-9468), humanized version 7D29 antibody,humanized version 7D19 antibody, humanized version 7D47 antibody,humanized version 7D39 antibody, humanized version 7D66 antibody,humanized version 8G9 antibody, humanized version 8G3 antibody,humanized version 8G4 antibody, humanized version 8G51 antibody,humanized version 8G22 antibody, humanized version 8G30 antibody,humanized version 8G46 antibody, humanized version 2A4 antibody (ATCCAccession Number PTA-9662), humanized version 2A20 antibody, humanizedversion 2A44 antibody, humanized version 2A77 antibody, humanizedversion 2A13 antibody, and humanized version 2A14 antibody. Hybridomasthat produce the 7D8 antibody (JH80 7D8.29.19.47) and the 2A4 antibody(JH80 2A4.20.44077) were deposited on Sep. 4, 2008, and on Dec. 17,2008, respectively, with the American Type Culture Collection (ATCC),currently located at 10801 University Boulevard, Manassas, Va.20110-2209, under the provisions of the Budapest Treaty for theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure (“Budapest Treaty”). The ATCC has assignedthe hybridoma producing 7D8 ATCC Accession No. PTA-9468, and thehybridoma producing 2A4 ATCC Accession No. PTA-9662.

Human isotype IgG1 is preferred for antibodies to the C terminal regionof AA because of it having highest affinity of human isotypes for theFcRI receptor on phagocytic cells. Some antibodies specifically bind toAA with a binding affinity greater than or equal to about 10⁷, 10⁸, 10⁹,or 10¹⁰ M.

Active immunization with fragments of AA can be combined with passiveadministration of antibodies. Examples of specific combinations includeAA fragments comprising HAA1 alpha isoform residues 70-76 withantibodies that specifically bind to epitope within HAA1 alpha isoformresidues 70-76; AA fragments comprising HAA1 alpha isoform residues70-76 with antibodies that specifically bind to epitope within HAA1alpha isoform residues 71-76; AA fragments comprising HAA1 alpha isoformresidues 70-76 with antibodies that specifically bind to epitope withinHAA1 alpha isoform residues 72-76; AA fragments comprising HAA1 alphaisoform residues 70-76 with antibodies that specifically bind to epitopewithin HAA1 alpha isoform residues 73-76; AA fragments comprising HAA1alpha isoform residues 70-76 with antibodies that specifically bind toepitope within HAA1 alpha isoform residues 74-76; AA fragmentscomprising HAA1 alpha isoform residues 70-76 with antibodies thatspecifically bind to epitope within HAA1 alpha isoform residues 70-75;AA fragments comprising HAA1 alpha isoform residues 70-76 withantibodies that specifically bind to epitope within HAA1 alpha isoformresidues 70-74; AA fragments comprising HAA1 alpha isoform residues70-76 with antibodies that specifically bind to epitope within HAA1alpha isoform residues 70-73; AA fragments comprising HAA1 alpha isoformresidues 70-76 with antibodies that specifically bind to epitope withinHAA1 alpha isoform residues 70-72; AA fragments comprising HAA1 alphaisoform residues 70-76 with antibodies that specifically bind to epitopewithin HAA1 alpha isoform residues 71-75; AA fragments comprising HAA1alpha isoform residues 70-76 with antibodies that specifically bind toepitope within HAA1 alpha isoform residues 72-75; AA fragmentscomprising HAA1 alpha isoform residues 70-76 with antibodies thatspecifically bind to epitope within HAA1 alpha isoform residues 73-75;AA fragments comprising HAA1 alpha isoform residues 70-76 withantibodies that specifically bind to epitope within HAA1 alpha isoformresidues 73-75; AA fragments comprising HAA1 alpha isoform residues70-76 with antibodies that specifically bind to epitope within HAA1alpha isoform residues 71-74; AA fragments comprising HAA1 alpha isoformresidues 70-76 with antibodies that specifically bind to epitope withinHAA1 alpha isoform residues 71-73; AA fragments comprising HAA1 alphaisoform residues 70-76 with antibodies that specifically bind to epitopewithin HAA1 alpha isoform residues 72-74. Additionally, AA fragmentscomprising HAA1 alpha isoform residues 71-76, 72-76, 73-76, 74-76,70-75, 70-74, 70-73, 70-72, 71-75, 72-75, 73-75, 71-74, 71-73, 72-74 maybe combined with antibodies that specifically bind to an epitope withinHAA1 alpha isoform residues 71-76, 72-76, 73-76, 74-76, 70-75, 70-74,70-73, 70-72, 71-75, 72-75, 73-75, 71-74, 71-73, 72-74. AA fragmentscomprising HAA1 alpha isoform residues 70-76, HAA1 beta isoform residues70-76, HAA1 gamma isoform residue 70-76, HAA2 alpha and beta isoformsresidues 70-76, MAA1 residues 69-75, MAA2 residues 69-75, or MAA3residues 62-68 may be combined with antibodies that specifically bind toan epitope within HAA1 alpha isoform residues 70-76, HAA1 beta isoformresidues 70-76, HAA1 gamma isoform residue 70-76, HAA2 alpha and betaisoforms residues 70-76, MAA1 residues 69-75, MAA2 residues 69-75, orMAA3 residues 62-68.

Some of the antibodies described above do not specifically bind themonomeric or precursor form of the amyloid protein. Some of suchantibodies specifically bind to a neoepitope generated upon cleavage ofa precursor protein resulting in an amyloid protein. For example, someantibodies specifically bind to the C-terminal residues of mouse AAfibrils -HEDT, (SEQ ID NO: 12), but do not specifically bind to apeptide that extends into the non-amyloid portion of SAA (GHEDTMADQE,SEQ ID NO: 61). Some antibodies specifically bind to a conformationalepitope. Some of such conformational epitopes are linear. Some of suchconformational epitopes are exposed when an amyloid protein enters anaggregated (e.g., fibrillar) structure or becomes partially denatured.Examples of such antibodies include murine monoclonal antibodies 2A4(ATCC Accession Number PTA-9662) and 7D8 (ATCC Accession NumberPTA-9468), human, humanized and chimeric forms thereof, other antibodiesthat specifically bind to the same epitope as 2A4, 8G9 or 7D8, andantigen-binding fragments of any such antibodies. Some antibodiesspecifically bind to an amyloid protein comprising the amino acidsequence ED. Some antibodies specifically bind to an amyloid proteinselected from the group consisting of immunoglobulin light chainprotein, human islet amyloid precursor polypeptide (IAPP), beta amyloidpeptide, transthyretin (TTR) and ApoA1.

The basic antibody structural unit is known to comprise a tetramer ofsubunits. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The amino-terminal portion of eachchain includes a variable region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The carboxy-terminalportion of each chain defines a constant region primarily responsiblefor effector function.

1. Antibodies

The invention includes intact antibodies and antigen-binding antibodyfragments, as well as pegylated antibodies and antibody fragments, aswell as antibodies with altered (e.g., reduced or eliminated) effectorfunction, for example, antibodies comprising mutations or substitutedresidues in the Fc region. Examples of immunologically active portionsof immunoglobulin molecules include F(ab) and F(ab′)2 tri-Fab′, Fab′,Fv, scFv, di-Fab′ fragments which can be generated by treating theantibody with an enzyme such as pepsin or produced by art-recognizedrecombinant engineering techniques. Additional antigen-binding fragmentsof antibodies of the invention include therapeutic antibody fragments,including pegylated antibody fragments, such as PEGylated Fab′ andPEGylated di-Fab′. Examples of effector function mutants are describedin U.S. Pat. No. 5,624,821, which is incorporated by reference herein inits entirety. Some antibodies have reduced binding affinity for Fc gammaRI receptor. Effector function mutant antibodies include antibodiescomprising mutations in the hinge region. Some mutant IgG antibodiescomprise a mutation in the heavy chain constant region at one or more ofpositions 234, 235, 236, 237, 297, 318, 320 and 322. In some antibodiesone or more of residues 234, 236 and 237 are substituted with alanine 1nsome antibodies, residue 235 is substituted with glutamine. In someantibodies, residue 297 is substituted with alanine 1n some antibodies,residues 318, 320 and 322 are substituted with alanine 1n someantibodies, residue 318 is substituted with valine. In some antibodies,residue 322 is substituted with glutamine. Antibodies with enhancedeffector function include antibodies single S239D and I332E and thedouble and triple mutants S239D/1332E and S239D/1332E/A330L (Kabatnumbering).

2. Polyclonal Antibodies

Polyclonal antibodies can be prepared as described above by immunizing asuitable subject with an immunogen. The antibody titer in the immunizedsubject can be monitored over time by standard techniques, such as withan enzyme linked immunosorbent assay (ELISA) using immobilized targetantigen. If desired, the antibody molecules directed against the targetantigen can be isolated from the mammal (e.g., from the blood) andfurther purified by well known techniques, such as protein A Sepharosechromatography to obtain the antibody, e.g., IgG, fraction. At anappropriate time after immunization, e.g., when the anti-antigenantibody titers are highest, antibody-producing cells can be obtainedfrom the subject and used to prepare monoclonal antibodies by standardtechniques, such as the hybridoma technique originally described byKohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al.(1981) J. Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem.255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. USA 76:2927-31;and Yeh et al. (1982) Int. J. Cancer 29:269-75). For the preparation ofchimeric polyclonal antibodies, see Buechler et al. U.S. Pat. No.6,420,113.

3. Monoclonal Antibodies

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating amonoclonal antibody (see, e.g., G. Galfre et al. (1977) Nature266:55052; Gefter et al. Somatic Cell Genet., cited supra; Lerner, YaleJ. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies, citedsupra). Moreover, the ordinarily skilled worker will appreciate thatthere are many variations of such methods which also would be useful.Typically, the immortal cell line (e.g., a myeloma cell line) is derivedfrom the same mammalian species as the lymphocytes. For example, murinehybridomas can be made by fusing lymphocytes from a mouse immunized withan immunogenic preparation of the present invention with an immortalizedmouse cell line. Preferred immortal cell lines are mouse myeloma celllines that are sensitive to culture medium containing hypoxanthine,aminopterin and thymidine (“HAT medium”). Any of a number of myelomacell lines can be used as a fusion partner according to standardtechniques, e.g., the P3-NS1/1-Ag4-1, P3-X63-Ag8.653 or Sp2/O-Ag14myeloma lines. These myeloma lines are available from ATCC. Typically,HAT-sensitive mouse myeloma cells are fused to mouse splenocytes usingpolyethylene glycol (“PEG”). Hybridoma cells resulting from the fusionare then selected using HAT medium, which kills unfused andunproductively fused myeloma cells (unfused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bind atarget antigen, e.g., Aβ, using a standard ELISA assay.

4. Recombinant Antibodies

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal antibody can be identified and isolated by screening arecombinant combinatorial immunoglobulin library (e.g., an antibodyphage display library) with a target antigen to thereby isolateimmunoglobulin library members that bind the target antigen. Kits forgenerating and screening phage display libraries are commerciallyavailable (e.g., the Pharmacia Recombinant Phage Antibody System,Catalog No. 27-9400-01; and the Stratagene SurfZAP™ Phage Display Kit,Catalog No. 240612). Additionally, examples of methods and reagentsparticularly amenable for use in generating and screening antibodydisplay library can be found in, for example, Ladner et al. U.S. Pat.No. 5,223,409; Kang et al. PCT International Publication No. WO92/18619; Dower et al. PCT International Publication No. WO 91/17271;Winter et al. PCT International Publication WO 92/20791; Markland et al.PCT International Publication No. WO 92/15679; Breitling et al. PCTInternational Publication WO 93/01288; McCafferty et al. PCTInternational Publication No. WO 92/01047; Garrard et al. PCTInternational Publication No. WO 92/09690; Ladner et al. PCTInternational Publication No. WO 90/02809; Fuchs et al. (1991)Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al.(1993) EMBO J. 12:725-734; Hawkins et al. (1992) J. Mol. Biol.226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gram et al.(1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad et al. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc. Acid Res.19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554.

5. Chimeric and Humanized Antibodies

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are withinthe scope of the invention.

The term “humanized immunoglobulin” or “humanized antibody” refers to animmunoglobulin or antibody that includes at least one humanizedimmunoglobulin or antibody chain (i.e., at least one humanized light orheavy chain). The term “humanized immunoglobulin chain” or “humanizedantibody chain” (i.e., a “humanized immunoglobulin light chain” or“humanized immunoglobulin heavy chain”) refers to an immunoglobulin orantibody chain (i.e., a light or heavy chain, respectively) having avariable region that includes a variable framework region substantiallyfrom a human immunoglobulin or antibody and complementarity determiningregions (CDRs) (e.g., at least one CDR, preferably two CDRs, morepreferably three CDRs) substantially from a non-human immunoglobulin orantibody, and further includes constant regions (e.g., at least oneconstant region or portion thereof, in the case of a light chain, andthree constant regions in the case of a heavy chain). The term“humanized variable region” (e.g., “humanized light chain variableregion” or “humanized heavy chain variable region”) refers to a variableregion that includes a variable framework region substantially from ahuman immunoglobulin or antibody and complementarity determining regions(CDRs) substantially from a non-human immunoglobulin or antibody.

The phrase “substantially from a human immunoglobulin or antibody” or“substantially human” means that, when aligned to a human immunoglobulinor antibody amino sequence for comparison purposes, the region shares atleast 80-90%, 90-95%, or 95-99% identity (i.e., local sequence identity)with the human framework or constant region sequence, allowing, forexample, for conservative substitutions, consensus sequencesubstitutions, germline substitutions, backmutations, and the like. Theintroduction of conservative substitutions, consensus sequencesubstitutions, germline substitutions, backmutations, and the like, isoften referred to as “optimization” of a humanized antibody or chain.The phrase “substantially from a non-human immunoglobulin or antibody”or “substantially non-human” means having an immunoglobulin or antibodysequence at least 80-95%, preferably at least 90-95%, more preferably,96%, 97%, 98%, or 99% identical to that of a non-human organism, e.g., anon-human mammal.

Accordingly, all regions or residues of a humanized immunoglobulin orantibody, or of a humanized immunoglobulin or antibody chain, except theCDRs, are substantially identical to the corresponding regions orresidues of one or more native human immunoglobulin sequences. The term“corresponding region” or “corresponding residue” refers to a region orresidue on a second amino acid or nucleotide sequence which occupies thesame (i.e., equivalent) position as a region or residue on a first aminoacid or nucleotide sequence, when the first and second sequences areoptimally aligned for comparison purposes.

The term “significant identity” means that two polypeptide sequences,when optimally aligned, such as by the programs GAP or BESTFIT usingdefault gap weights, share at least 50-60% sequence identity, preferablyat least 60-70% sequence identity, more preferably at least 70-80%sequence identity, more preferably at least 80-90% sequence identity,even more preferably at least 90-95% sequence identity, and even morepreferably at least 95% sequence identity or more (e.g., 99% sequenceidentity or more). The term “substantial identity” means that twopolypeptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80-90% sequenceidentity, preferably at least 90-95% sequence identity, and morepreferably at least 95% sequence identity or more (e.g., 99% sequenceidentity or more). For sequence comparison, typically one sequence actsas a reference sequence, to which test sequences are compared. Whenusing a sequence comparison algorithm, test and reference sequences areinput into a computer, subsequence coordinates are designated, ifnecessary, and sequence algorithm program parameters are designated. Thesequence comparison algorithm then calculates the percent sequenceidentity for the test sequence(s) relative to the reference sequence,based on the designated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., Current Protocols in Molecular Biology). One example ofalgorithm that is suitable for determining percent sequence identity andsequence similarity is the BLAST algorithm, which is described inAltschul et al., J. Mol. Biol. 215:403 (1990). Software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information (publicly accessible through the NationalInstitutes of Health NCBI internet server). Typically, default programparameters can be used to perform the sequence comparison, althoughcustomized parameters can also be used. For amino acid sequences, theBLASTP program uses as defaults a wordlength (W) of 3, an expectation(E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff,Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

Preferably, residue positions which are not identical differ byconservative amino acid substitutions. For purposes of classifying aminoacids substitutions as conservative or nonconservative, amino acids aregrouped as follows: Group I (hydrophobic sidechains): leu, met, ala,val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser,thr; Group III (acidic side chains): asp, glu; Group IV (basic sidechains): asn, gln, his, lys, arg; Group V (residues influencing chainorientation): gly, pro; and Group VI (aromatic side chains): trp, tyr,phe. Conservative substitutions involve substitutions between aminoacids in the same class. Non-conservative substitutions constituteexchanging a member of one of these classes for a member of another.

Preferably, humanized immunoglobulins or antibodies bind antigen with anaffinity that is within a factor of three, four, or five of that of thecorresponding non-humanized antibody. For example, if the nonhumanizedantibody has a binding affinity of 10⁻⁹ M, humanized antibodies willhave a binding affinity of at least 3×10⁻⁸ M, 4×10⁻⁸ M, 5×10⁻⁸ M, or10⁻⁹ M. When describing the binding properties of an immunoglobulin orantibody chain, the chain can be described based on its ability to“direct antigen (e.g., Aβ) binding”. A chain is said to “direct antigenbinding” when it confers upon an intact immunoglobulin or antibody (orantigen binding fragment thereof) a specific binding property or bindingaffinity. A mutation (e.g., a backmutation) is said to substantiallyaffect the ability of a heavy or light chain to direct antigen bindingif it affects (e.g., decreases) the binding affinity of an intactimmunoglobulin or antibody (or antigen binding fragment thereof)comprising said chain by at least an order of magnitude compared to thatof the antibody (or antigen binding fragment thereof) comprising anequivalent chain lacking said mutation. A mutation “does notsubstantially affect (e.g., decrease) the ability of a chain to directantigen binding” if it affects (e.g., decreases) the binding affinity ofan intact immunoglobulin or antibody (or antigen binding fragmentthereof) comprising said chain by only a factor of two, three, or fourof that of the antibody (or antigen binding fragment thereof) comprisingan equivalent chain lacking said mutation.

The term “chimeric immunoglobulin” or antibody refers to animmunoglobulin or antibody whose variable regions derive from a firstspecies and whose constant regions derive from a second species.Chimeric immunoglobulins or antibodies can be constructed, for exampleby genetic engineering, from immunoglobulin gene segments belonging todifferent species. The terms “humanized immunoglobulin” or “humanizedantibody” are not intended to encompass chimeric immunoglobulins orantibodies, as defined infra. Although humanized immunoglobulins orantibodies are chimeric in their construction (i.e., comprise regionsfrom more than one species of protein), they include additional features(i.e., variable regions comprising donor CDR residues and acceptorframework residues) not found in chimeric immunoglobulins or antibodies,as defined herein.

Such chimeric and humanized monoclonal antibodies can be produced byrecombinant DNA techniques known in the art, for example using methodsdescribed in Robinson et al. International Application No.PCT/US86/02269; Akira, et al. European Patent Application 184,187;Taniguchi, M., European Patent Application 171,496; Morrison et al.European Patent Application 173,494; Neuberger et al. PCT InternationalPublication No. WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567;Cabilly et al. European Patent Application 125,023; Better et al. (1988)Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al.(1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987)Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shawet al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S. L.(1985) Science 229:1202-1207; Oi et al. (1986) BioTechniques 4:214;Winter U.S. Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525;Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J.Immunol. 141:4053-4060. Therapeutic agents also include antibodymimetics such as complementarity determining region (CDR) mimetics.

6. Human Antibodies from Transgenic Animals and Phage Display

Alternatively, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant miceresults in the production of human antibodies upon antigen challenge.See, e.g., U.S. Pat. Nos. 6,150,584; 6,114,598; and 5,770,429.

Fully human antibodies can also be derived from phage-display libraries(Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol.Biol., 222:581-597 (1991)). Chimeric polyclonal antibodies can also beobtained from phage display libraries (Buechler et al. U.S. Pat. No.6,420,113).

7. Bispecific Antibodies, Antibody Fusion Polypeptides, and Single-ChainAntibodies

Bispecific antibodies (BsAbs) are antibodies that have bindingspecificities for at least two different epitopes. Such antibodies canbe derived from full length antibodies or antibody fragments (e.g.F(ab)′2 bispecific antibodies). Methods for making bispecific antibodiesare known in the art. Traditional production of full length bispecificantibodies is based on the coexpression of two immunoglobulin heavychain-light chain pairs, where the two chains have differentspecificities (Millstein et al., Nature, 305:537-539 (1983)). Because ofthe random assortment of immunoglobulin heavy and light chains, thesehybridomas (quadromas) produce a potential mixture of different antibodymolecules (see, WO 93/08829 and in Traunecker et al., EMBO J.,10:3655-3659 (1991)).

Bispecific antibodies also include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin or other payload.Heteroconjugate antibodies may be made using any convenientcross-linking methods. Suitable cross-linking agents are well known inthe art, and are disclosed in U.S. Pat. No. 4,676,980, along with anumber of cross-linking techniques.

In yet another aspect, the antibody can be fused, chemically orgenetically, to a payload such as a reactive, detectable, or functionalmoiety, for example, an immunotoxin to produce an antibody fusionpolypeptide. Such payloads include, for example, immunotoxins,chemotherapeutics, and radioisotopes, all of which are well-known in theart.

Single chain antibodies are also suitable for stabilization according tothe invention. The fragments comprise a heavy-chain variable domain (VH)connected to a light-chain variable domain (VL) with a linker, whichallows each variable region to interface with each other and recreatethe antigen binding pocket of the parent antibody from which the VL andVH regions are derived. See Gruber et al., J. Immunol., 152:5368 (1994).

It is understood that any of the foregoing polypeptide molecules, aloneor in combination, are suitable for preparation as stabilizedformulations according to the invention.

XI. Subjects Amenable to Treatment

Subjects or patients amenable to treatment include individuals at riskof disease but not showing symptoms, as well as patients presentlyshowing symptoms. Therefore, the present methods can be administeredprophylactically to the general population without the need for anyassessment of the risk of the subject patient. The present methods areespecially useful for individuals who do have a known genetic riskautoimmune disorders. Such individuals include those having relativeswho have experienced this disease and those whose risk is determined byanalysis of genetic or biochemical markers.

Patients suffering from AA amyloidosis can be asymptomatic for aprolonged period of time. Therefore, clinical diagnosis of AAamyloidosis is often delayed or missed until the amyloid deposits areextensive. For those patients who are symptomatic, it is estimated thatonly 53% of the cases are diagnosed. See L.E.K. Consulting, IndependentMarket Research (2003).

The invention provides methods useful to treat or effect prophylaxis ofa disease characterized by the deposition of an amyloid protein, suchas, for example, the diseases described above, including those listed inTable 1. Some methods are useful to treat or effect prophylaxis of adisease characterized by the deposition of an amyloid protein comprisingthe amino acid sequence ED. In some methods, if the amyloid proteincomprises the amino acid sequence AEDV, then the antibody is notadministered to treat or effect prophylaxis of Alzheimer's disease orMild Cognitive Impairment. The amyloid protein can be any of the amyloidproteins described above, including those listed in Table 1, such as,for example, serum amyloid A protein, immunoglobulin light chainprotein, such as, for example, Vλ6 Wil or Vκ, human islet amyloidprecursor polypeptide (IAPP), beta amyloid peptide, transthyretin (TTR)or ApoA1.

The present methods are especially useful for individuals who do have aknown risk of, are suspected to have, or have been diagnosed with AAamyloidosis or AL amyloidosis. Such individuals include but are notlimited to those having chronic inflammatory diseases, inheritedinflammatory diseases, and chronic microbial infections, such asrheumatoid arthritis, juvenile chronic arthritis, ankylosingspondylitis, psoriasis, psoriatic arthropathy, Reiter's syndrome, AdultStill's disease, Behcet's syndrome, Crohn's disease, FamilialMediterranean Fever, leprosy, tuberculosis, bronchiectasis, decubitusulcers, chronic pyelonephritis, osteomyelitis, Whipple's disease,myeloma, macroglobulinemia, immunocyte dyscrasia, monoclonal gammopathy,occult dyscrasia. Chronic inflammatory and infectious conditions areprerequisite to the development of AA amyloidosis and AL amyloidosismanifested by local nodular amyloidosis can be associated with chronicinflammatory diseases. Individuals who do have known risk of AAamyloidosis also include but are not limited to those having malignantneoplasms as Hodgkin's lymphoma, renal carcinoma, carcinomas of gut,lung and urogenital tract, basal cell carcinoma, and hairy cellleukemia. Additionally, individuals who do have known risk of AAamyloidosis also include but are not limited to those havinglymphoproliferative disorders such as Castleman's Disease.

In both asymptomatic and symptomatic patients, treatment can begin atany time before or after the diagnosis of the underlying AA or ALamyloid diseases. Treatment typically entails multiple dosages over aperiod of time. Treatment can be monitored by assaying antibody,activated T-cell (a side effect) or B-cell responses to the therapeuticagent (e.g., AA peptide), or employing radiolabeled SAP Scintigraphyover time. If the response falls, a booster dosage is indicated.

XII. Treatment Regimes

In general, treatment regimes involve administering an agent effectiveto induce an immunogenic response to an amyloid protein, and preferablyto an aggregated form of such amyloid protein, such as, for example, AAor AL. Preferably an immunogenic fragment of AA or AL or an X₁EDX₂fragment is administered to a patient. In prophylactic applications,pharmaceutical compositions or medicaments are administered to a patientsusceptible to, or otherwise at risk of, amyloidosis such as AAAmyloidosis or AL amyloidosis, in an amount sufficient to eliminate orreduce the risk, lessen the severity, or delay the onset of the disease,including physiological, biochemical, histologic and/or behavioralsymptoms of the disease, its complications and intermediate pathologicalphenotypes presenting during development of the disease. In therapeuticapplications, an agent is administered to a patient suspected of, oralready suffering from such a disease in a regime comprising an amountand frequency of administration of the agent sufficient to cure, or atleast partially arrest, or inhibit deterioration of the symptoms of thedisease (physiological, biochemical, histologic and/or behavioral),including its complications and intermediate pathological phenotypes indevelopment of the disease. In some methods, administration of agentreduces or eliminates early symptomology in patients that have not yetdeveloped characteristic AA or AL Amyloidosis pathology. An amountadequate to accomplish therapeutic or prophylactic treatment is definedas a therapeutically- or prophylactically-effective dose. A combinationof amount and dosage frequency adequate to accomplish the therapeutic orprophylactic treatment is defined as a therapeutically- orprophylactically-effective regime. In both prophylactic and therapeuticregimes, agents are usually administered in several dosages until asufficient immune response has been achieved. A dosage and frequency ofadministrations adequate to accomplish therapeutic or prophylactictreatment is defined as a therapeutically- or prophylactically-effectiveregime. Typically, the patient's immune response is monitored andrepeated dosages are given if the immune response starts to wane. Theimmune response can be monitored by detecting antibodies, for example,to AA or AL in the blood in the patient or detecting levels of, forexample, AA or AL.

Effective doses of the agents and compositions of the present invention,for the treatment of the above described conditions vary depending uponmany different factors, including means of administration, target site,physiological state of the patient, whether the patient is human or ananimal, other medications administered, and whether treatment isprophylactic or therapeutic. Usually, the patient is a human butnonhuman mammals including transgenic mammals can also be treated.Treatment dosages need to be titrated to optimize safety and efficacy.The amount of immunogen depends on whether adjuvant is alsoadministered, with higher dosages being required in the absence ofadjuvant. The amount of an immunogen for administration sometimes variesfrom 1-500 μg per patient and more usually from 5-500 μg per injectionfor human administration. Occasionally, a higher dose of 1-2 mg perinjection is used. Typically at least 10, 20, 50 or 100 μg is used foreach human injection. The mass of immunogen also depends on the massratio of immunogenic epitope within the immunogen to the mass ofimmunogen as a whole. Typically, 10⁻³ to 10⁻⁵ micromoles of immunogenicepitope are used for microgram of immunogen. The timing of injectionscan vary significantly from once a day, to once a year, to once adecade. On any given day that a dosage of immunogen is given, the dosageis greater than 1 μg/patient and usually greater than 10 μg/patient ifadjuvant is also administered, and greater than 10 μg/patient andusually greater than 100 μg/patient in the absence of adjuvant. Atypical regimen consists of an immunization followed by boosterinjections at time intervals, such as 6 week intervals. Another regimenconsists of an immunization followed by booster injections 1, 2 and 12months later. Another regimen entails an injection every two months forlife. Alternatively, booster injections can be on an irregular basis asindicated by monitoring of immune response.

Doses for nucleic acids encoding immunogens range from about 10 ng to 1g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg DNA per patient. Dosesfor infectious viral vectors vary from 10-100, or more, virions perdose.

For passive immunization with an antibody (in combination therapies),the dosage ranges from about 0.0001 to 100 mg/kg, 0.5 to less than 5mg/kg, and more usually 0.01 to 5 mg/kg, 0.5 to 3 mg/kg, of the hostbody weight. For example dosages can be 1 mg/kg body weight or 10 mg/kgbody weight or within the range of 1-10 mg/kg or in other words, 70 mgor 700 mg or within the range of 70-700 mg, respectively, for a 70 kgpatient. As an additional example, dosages can be less than 5 mg/kg bodyweight or 1.5 mg/kg body weight or within the range of 0.5 to 1.5 mg/kg,preferably at least 1.5 mg/kg. An exemplary treatment regime entailsadministration once per every two weeks or once a month or once every 3to 6 months. In some methods, two or more monoclonal antibodies withdifferent binding specificities are administered simultaneously, inwhich case the dosage of each antibody administered falls within theranges indicated. Antibody is usually administered on multipleoccasions. Intervals between single dosages can be weekly, monthly oryearly. Intervals can also be irregular as indicated by measuring bloodlevels of antibody to AA in the patient. In some methods, dosage isadjusted to achieve a plasma antibody concentration of 1-1000 μg/ml andin some methods 25-300 μg/ml. Alternatively, antibody can beadministered as a sustained release formulation, in which case lessfrequent administration is required. Dosage and frequency vary dependingon the half-life of the antibody in the patient. In general, humanantibodies show the longest half life, followed by humanized antibodies,chimeric antibodies, and nonhuman antibodies. The dosage and frequencyof administration can vary depending on whether the treatment isprophylactic or therapeutic. In prophylactic applications, a relativelylow dosage is administered at relatively infrequent intervals over along period of time. Some patients continue to receive treatment for therest of their lives. In therapeutic applications, a relatively highdosage at relatively short intervals is sometimes required untilprogression of the disease is reduced or terminated, and preferablyuntil the patient shows partial or complete amelioration of symptoms ofdisease. Thereafter, the patent can be administered a prophylacticregime.

Agents for inducing an immune response can be administered byparenteral, topical, intravenous, oral, subcutaneous, intraarterial,intracranial, intraperitoneal, intranasal or intramuscular means forprophylactic and/or therapeutic treatment. The most typical route ofadministration of an immunogenic agent is subcutaneous although otherroutes can be equally effective. The next most common route isintramuscular injection. This type of injection is most typicallyperformed in the arm or leg muscles. In some methods, agents areinjected directly into a particular tissue where deposits haveaccumulated, e.g., intracranial injection. Intramuscular injection orintravenous infusion is preferred for administration of antibody (incombination therapies). In some methods, particular therapeuticantibodies are injected directly into the cranium. In some methods,antibodies are administered as a sustained release composition ordevice, such as a MEDIPAD™ device.

Agents of the invention are often administered as pharmaceuticalcompositions comprising an active therapeutic agent, i.e., and a varietyof other pharmaceutically acceptable components. See Remington'sPharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa.,1980). The preferred form depends on the intended mode of administrationand therapeutic application. The compositions can also include,depending on the formulation desired, pharmaceutically-acceptable,non-toxic carriers or diluents, which are defined as vehicles commonlyused to formulate pharmaceutical compositions for animal or humanadministration. The diluent is selected so as not to affect thebiological activity of the combination. Examples of such diluents aredistilled water, physiological phosphate-buffered saline, Ringer'ssolutions, dextrose solution, and Hank's solution. In addition, thepharmaceutical composition or formulation may also include othercarriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenicstabilizers and the like.

Pharmaceutical compositions can also include large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such as latexfunctionalized SEPHAROSE™, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (such as oildroplets or liposomes). Additionally, these carriers can function asimmuno stimulating agents (i.e., adjuvants).

For parenteral administration, agents of the invention can beadministered as injectable dosages of a solution or suspension of thesubstance in a physiologically acceptable diluent with a pharmaceuticalcarrier that can be a sterile liquid such as water oils, saline,glycerol, or ethanol. Additionally, auxiliary substances, such aswetting or emulsifying agents, surfactants, pH buffering substances andthe like can be present in compositions. Other components ofpharmaceutical compositions are those of petroleum, animal, vegetable,or synthetic origin, for example, peanut oil, soybean oil, and mineraloil. In general, glycols such as propylene glycol or polyethylene glycolare preferred liquid carriers, particularly for injectable solutions.Antibodies can be administered in the form of a depot injection orimplant preparation which can be formulated in such a manner as topermit a sustained release of the active ingredient. An exemplarycomposition comprises monoclonal antibody at 5 mg/mL, formulated inaqueous buffer consisting of 50 mM L-histidine, 150 mM NaCl, adjusted topH 6.0 with HCl. Compositions for parenteral administration aretypically substantially sterile, isotonic and manufactured under GMPconditions of the FDA or similar body.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect, as discussed above (see Langer, Science 249,1527 (1990) and Hanes, Advanced Drug Delivery Reviews 28, 97-119 (1997).The agents of this invention can be administered in the form of a depotinjection or implant preparation which can be formulated in such amanner as to permit a sustained or pulsatile release of the activeingredient.

Additional formulations suitable for other modes of administrationinclude oral, intranasal, and pulmonary formulations, suppositories, andtransdermal applications.

For suppositories, binders and carriers include, for example,polyalkylene glycols or triglycerides; such suppositories can be formedfrom mixtures containing the active ingredient in the range of 0.5% to10%, preferably 1%-2%. Oral formulations include excipients, such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, and magnesium carbonate. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders and contain 10%-95%of active ingredient, preferably 25%-70%.

Topical application can result in transdermal or intradermal delivery.Topical administration can be facilitated by co-administration of theagent with cholera toxin or detoxified derivatives or subunits thereofor other similar bacterial toxins (See Glenn et al., Nature 391, 851(1998)). Co-administration can be achieved by using the components as amixture or as linked molecules obtained by chemical crosslinking orexpression as a fusion protein.

Alternatively, transdermal delivery can be achieved using a skin path orusing transferosomes (Paul et al., Eur. J. Immunol. 25, 3521-24 (1995);Cevc et al., Biochem. Biophys. Acta 1368, 201-15 (1998)).

XIII. Combinational Drug Therapy Treatment Regimes

Combination therapy according to the invention may be performed alone orin conjunction with another therapy to treat or effect prophylaxis of AAamyloidosis. Combination therapy according to the invention may also beperformed in conjunction with another therapy which treats or effectsprophylaxis of an underlying amyloid disease such as inflammatorydiseases, chronic microbial infections, malignant neoplasms, inheritedinflammatory diseases, and lymphoproliferative disorders. There arelarge numbers of treatments available in commercial use, in clinicalevaluation and in pre-clinical development, which could be selected foruse with the presently disclosed invention for effecting prophylaxis andtreatment of AA amyloidosis by combination drug therapy. Such treatmentscan be one or more compounds selected from, but not limited to severalmajor categories, namely, (i) non-steroidal anti-inflammatory drugs(NSAIDs; e.g., detoprofen, diclofenac, diflunisal, etodolac, fenoprofen,flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenameate,mefenamic acid, meloxicam, nabumeone, naproxen sodium, oxaprozin,piroxicam, sulindac, tolmetin, celecoxib, rofecoxib, aspirin, cholinesalicylate, salsalte, and sodium and magnesium salicylate); (ii)steroids (e.g., cortisone, dexamethasone, hydrocortisone,methylprednisolone, prednisolone, prednisone, triamcinolone); (iii)DMARDs, i.e., disease modifying antirheumatic drugs (e.g., cyclosporine,azathioprine, methotrexate, leflunomide, cyclophosphamide,hydroxychloroquine, sulfasalazine, D-penicillamine, minocycline, andgold); or (iv) recombinant proteins (e.g., ENBREL® (etanercept, asoluble TNF receptor) and REMICADE® (infliximab) a chimeric monoclonalanti-TNF antibody).

The duration of the combination therapy depends on the type ofunderlying disease being treated, the age and condition of the patient,the stage and type of the patient's disease, and how the patientresponds to the treatment. The doctor can observe the therapy's effectsclosely and make any adjustments that are needed. Additionally, a personhaving a greater risk of developing AA Amyloidosis (e.g., a person whois genetically predisposed or previously had an inflammatory disorder orother underlying diseases) or AL amyloidosis may receive prophylactictreatment to inhibit or delay the development of AA AL aggregates suchas fibrils.

The dosage, frequency and mode of administration of each component ofthe combination can be controlled independently. For example, onecompound may be administered orally three times per day, while thesecond compound may be administered intramuscularly once per day.Combination therapy may be given in on-and-off cycles that include restperiods. The compounds may also be formulated together such that oneadministration delivers both compounds. The combination of the inventioncan also be provided as components of a pharmaceutical pack. The drugscan be formulated together or separately and in individual dosageamounts. Each compound is admixed with a suitable carrier substance, andis generally present in an amount of 1-95% by weight of the total weightof the composition.

The composition may be provided in a dosage form that is suitable fororal, parenteral (e.g., intravenous, intramuscular, subcutaneous),rectal, transdermal, nasal, vaginal, inhalant, or ocular administration.Thus, the composition may be in form of, e.g., tablets, capsules, pills,powders, granulates, suspensions, emulsions, solutions, gels includinghydrogels, pastes, ointments, creams, plasters, drenches, deliverydevices, suppositories, enemas, injectables, implants, sprays, oraerosols. The pharmaceutical compositions may be formulated according toconventional pharmaceutical practice (see, e.g., Remington: The Scienceand Practice of Pharmacy, (19th ed.) ed. A. R. Gennaro, 1995, MackPublishing Company, Easton, Pa. and Encyclopedia of PharmaceuticalTechnology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, MarcelDekker, N.Y.

XIV. Methods of Monitoring or Diagnosing AA or AL Amyloidosis

Methods of monitoring or diagnosing AA or AL amyloidosis includemeasuring the plasma concentrations of SAA and C-reactive protein,performing tissue biopsy (renal, rectal, gastric, gingival, fat,salivary, labial glands) and histology with congo red staining and/orimmunostaining with specific antibodies directed against AA or ALaggregates such as fibrils. The invention provides methods of detectingan antibody response against AA peptide in a patient suffering from orsusceptible to AA Amyloidosis. The methods are particularly useful formonitoring a course of treatment being administered to a patient. Themethods can be used to monitor both therapeutic treatment on symptomaticpatients and prophylactic treatment on asymptomatic patients. Somemethods entail determining a baseline value of an antibody response in apatient before administering a dosage of an immunogenic agent, andcomparing this with a value for the immune response after treatment. Asignificant increase (i.e., greater than the typical margin ofexperimental error in repeat measurements of the same sample, expressedas one standard deviation from the mean of such measurements) in valueof the antibody response signals a positive treatment outcome (i.e.,that administration of the agent has achieved or augmented an immuneresponse). If the value for the antibody response does not changesignificantly, or decreases, a negative treatment outcome is indicated.In general, patients undergoing an initial course of treatment with animmunogenic agent are expected to show an increase in antibody responsewith successive dosages, which eventually reaches a plateau.Administration of agent is generally continued while the antibodyresponse is increasing. Attainment of the plateau is an indicator thatthe administered of treatment can be discontinued or reduced in dosageor frequency.

In other methods, a control value (i.e., a mean and standard deviation)of an antibody response is determined for a control population.Typically the individuals in the control population have not receivedprior treatment. Measured values of the antibody response in a patientafter administering a therapeutic agent are then compared with thecontrol value. A significant increase relative to the control value(e.g., greater than one standard deviation from the mean) signals apositive treatment outcome. A lack of significant increase or a decreasesignals a negative treatment outcome. Administration of agent isgenerally continued while the antibody response is increasing relativeto the control value. As before, attainment of a plateau relative tocontrol values in an indicator that the administration of treatment canbe discontinued or reduced in dosage or frequency.

In other methods, a control value of antibody response (e.g., a mean andstandard deviation) is determined from a control population ofindividuals who have undergone treatment with a therapeutic agent andwhose antibody responses have reached a plateau in response totreatment. Measured values of antibody response in a patient arecompared with the control value. If the measured level in a patient isnot significantly different (e.g., more than one standard deviation)from the control value, treatment can be discontinued. If the level in apatient is significantly below the control value, continuedadministration of agent is warranted. If the level in the patientpersists below the control value, then a change in treatment regime, forexample, use of a different adjuvant, fragment or switch to passiveadministration may be indicated.

In other methods, a patient who is not presently receiving treatment buthas undergone a previous course of treatment is monitored for antibodyresponse to determine whether a resumption of treatment is required. Themeasured value of antibody response in the patient can be compared witha value of antibody response previously achieved in the patient after aprevious course of treatment. A significant decrease relative to theprevious measurement (i.e., greater than a typical margin of error inrepeat measurements of the same sample) is an indication that treatmentcan be resumed. Alternatively, the value measured in a patient can becompared with a control value (mean plus standard deviation) determinedin a population of patients after undergoing a course of treatment.Alternatively, the measured value in a patient can be compared with acontrol value in populations of prophylactically treated patients whoremain free of symptoms of disease, or populations of therapeuticallytreated patients who show amelioration of disease characteristics. Inall of these cases, a significant decrease relative to the control level(i.e., more than a standard deviation) is an indicator that treatmentshould be resumed in a patient.

Some methods employ iodine-123-labeled or iodine-125-labeled serumamyloid P component (¹²³I-SAP or ¹²⁵I-SAP) Scintigraphy. ¹²³I-SAP or¹²⁵I-SAP is intravaneously injected into patients and viewed with gammacamera. Radiolabeled SAP Scintigraphy is a useful method to monitor theprogression of amyloidosis in patients and evaluate treatments. It isspecific for amyloid and can be used to quantitatively monitor thelocation and amount of amyloid deposits in patients. ¹²³I-SAP and¹²⁵I-SAP do not accumulate in healthy subjects or in non-amyloidpatients. Radiolabeled SAP scintigraphy can be used to monitor dynamicturnover of amyloid, and can assess the efficacy of treatments aimed atregressing amyloid deposits. Further, radiolabeled SAP Scintigraphy isnon-invasive and provides whole body scan. Methods of the inventionentail determining a baseline value of an antibody response in a patientbefore administering a dosage of an agent, and comparing this with avalue for the immune response after treatment in a patient. Asignificant increase (i.e., greater than the typical margin ofexperimental error in repeat measurements of the same sample, expressedas one standard deviation from the mean of such measurements) in valueof the antibody response signals a positive treatment outcome (i.e.,that administration of the agent has achieved or augmented an immuneresponse). If the value for the antibody response does not changesignificantly, or decreases, a negative treatment outcome is indicated.In general, patients undergoing an initial course of treatment with animmunogenic agent are expected to show an increase in antibody responsewith successive dosages, which eventually reaches a plateau.Administration of agent is generally continued while the antibodyresponse is increasing. Attainment of the plateau is an indicator thatthe administered of treatment can be discontinued or reduced in dosageor frequency.

The tissue sample for analysis is typically blood, plasma, serum, mucousor cerebrospinal fluid from the patient. The sample is analyzed forindication of an immune response to any form of AA or AL peptide. Theimmune response can be determined from the presence of antibodies thatspecifically bind to AA or AL peptide. Antibodies can be detected in abinding assay to a ligand that specifically binds to the antibodies.Typically the ligand is immobilized. Binding can be detected using alabeled anti-idiotypic antibody.

In combination regimes employing both active and passive administration,analogous approaches can be used to monitor levels of antibody resultingfrom passive administration.

Methods of diagnosing amyloidosis can also be employed by, e.g.,administering to a subject an antibody or antigen-binding fragmentthereof, that is bound to a detectable label, wherein the antibody orfragment thereof specifically binds to an epitope including X₁EDX₂ in anaggregated amyloid protein, wherein X₁ and X₂ are any amino acids, anddetecting the presence or absence of the bound antibody or fragmentthereof. Detection of the bound antibody or fragment supports adiagnosis of amyloidosis. Antibodies and fragments useful in thediagnosis of amyloidosis include the disclosed antibodies of theinvention.

The diagnostic antibodies or fragments of the invention can beadministered, by e.g., intravenous injection into the body of a patient,or directly into the brain by intracranial injection. The antibodydosage is readily determined by one skilled in the art. Typically, theantibody is labeled, although in some methods, the antibody is unlabeledand a secondary labeling agent is used to bind to the antibody. Thechoice of label depends on the means of detection. For example, afluorescent label is suitable for optical detection. Use of paramagneticlabels is suitable for tomographic detection without surgicalintervention. Radiolabels may be used including ²¹¹At, ²¹²Bi, ⁶⁷Cu,¹²⁵I, ¹³¹I, ¹¹¹In, ³²P, ²¹²Pb, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ^(99m)Tc, or ⁹⁰Y.Such labels may be detected using PET or SPECT or other suitabletechnique.

Diagnosis may also be performed by comparing the number, size, and/orintensity of labeled loci, to corresponding baseline values. The baseline values can represent the mean levels in a population of undiseasedindividuals. Baseline values can also represent previous levelsdetermined in the same patient. For example, baseline values can bedetermined in a patient, and measured values thereafter compared withthe baseline values. An increase in values relative to baseline signalssupports a diagnosis of AA amyloidosis.

The diagnostic methods of the invention may be used to diagnoseamyloidosis diseases including AA amyloidosis, AL amyloidosis,Alzheimer's disease, Mild Cognitive Impairment, amyloid polyneuropathy,Mediterranean fever, Muckle-Wells syndrome, reactive systemicamyloidosis associated with systemic inflammatory diseases, myeloma ormacroglobulinemia associated amyloidosis, amyloidosis associated withimmunocyte dyscrasia, monoclonal gammopathy, occult dyscrasia, or localnodular amyloidosis associated with chronic inflammatory diseases.

XV. Animal Models of AA Amyloidosis

AA amyloidosis can be induced experimentally in mice in which SAAconcentrations are markedly increased by injection of silver nitrate,casein, or lipopolysaccharide. These agents stimulate the production ofcytokines See Skinner et al. Lab Invest. 36:420-427 (1997) andKisilevsky et al. Bailliere's Clin. Immunol. Immunopathol. 8(3) 613-626(1994). Within 2 or 3 weeks after the inflammatory stimulus, animalsdevelop systemic AA deposits, as found in patients with AA Amyloidosis.This lag phase is dramatically shortened when mice are given,concomitantly, an intravenous injection of protein extracted from AAamyloid-laden mouse spleen or liver. See Axelrad et al. Lab Invest.47(2):139-46 (1982). The amyloidogenic accelerating activity of suchpreparations was termed “amyloid enhancing factor” (AEF). Lundmark etal. reports that the active principle of AEF is unequivocally the AAfiber itself. Further, they demonstrated that this material is extremelypotent, being active in doses less than 1 ng, and that it retained itsbiologic activity over a considerable length of time. Notably, the AEFwas also effective when administered orally. They concluded that AA andperhaps other forms of amyloidosis are transmissible diseases, akin tothe prion-associated disorders. See Lundmark et al. Proc. Nat. Acad.Sci. 99: 6979-6984 (2002).

AA amyloid can also be induced in transgenic strains of mice carryingthe human interleukin 6 gene under the control of the metallothionein-Ipromoter resulting in markedly increased concentrations of SAA anddeveloping amyloid in the spleen, liver and kidneys by 3 months of age.At the time of death at about 8-9 months, organs from these transgenicmice have extensive amyloid deposits. See Solomon et al., Am. J. Pathol.154(4):1267-1272 (1999).

The Transgenic Rapidly Induced Amyloid Disease (TRIAD) transgenic mousemodel is an improvement to the above described transgenic mouse model.TRIAD mice carry the human interleukin 6 gene under the control of theH-2L^(D) histocompatibility promoter. Administration of AEF to 8-weekold TRIAD mice results in prominent spenic and hepatic AA amyloiddeposits within 3 to 4 weeks. Subsequently, this process progresses toother organs, leading to death 4-6 weeks later. The development of thesystemic amyloidosis is accelerated compared to the above-describedtransgenic mouse model. See University of Tennessee ResearchCorporation, WO 01/77167, Pharmacopeia, WO 95/35503 and Scripps, WO95/30642 Wall et al. Amyloid 12(3): 149-156 (2005) (each of which isincorporated by reference for all purposes).

The common marmoset (Callithrix jacchus) is a small New World primatenative to Brazil that has been used extensively in biomedical research.Ludlage et al. reports that common marmoset were found to have amyloiddeposits in one or more organs, including the liver, adrenal glands,kidneys, and intestine. The authors posit that hereditary factors mightbe responsible for the development of AA amyloidosis in this primate. Inthis regard, the common marmoset could serve as a unique experimentalmodel for study of the pathogenesis and therapy of AA and other systemicamyloid disorders. See Ludlage et al. Vet Pathol 42:117-124 (2005).

The Shar Pei species of dog, a breed having an AA sequence with the-AEDS motif and that is particularly susceptible to AA-amyloidosis,provides a naturally occurring model of systemic AA in which to evaluatenovel diagnostic and therapeutic applications of AA amyloid-specificantibodies and other compounds.

EXAMPLES Example I AA Fragments

Peptides corresponding to amino acids 71-75 -GHEDT, as described byYamamoto and Migita Proc. Natl. Acad. Sci. USA 82:2915-2919 weresynthesized by AnaSpec, San Jose, Calif., USA. Polyclonal antibodies(Pab) AA were raised and the immunoglobulin fraction isolated, aspreviously described by Bard, F. et al., (2000) Nat. Med. 6, 916-919.

Example II Immunogen for Preparation of Murine Antibodies

The epitope used was GHEDT, (SEQ ID NO: 3) with a CG linker at its Nterminus. The peptide EPRB-39 which contains the epitope is coupled tosheep anti mouse antibody. EPRB-39 is obtained from Anasec, San Jose,Calif. The antibodies produced appear to be neoepitope specific becausethey don't specifically bind to a peptide that spans the regionGHEDTIADQE, (SEQ ID NO: 89).

Example III Immunization Procedures

Six-week-old A/J mice were intraperitoneal injected with 50 ugEPRB-39/sheep anti-mouse IgG with Complete Freund's Adjuvant (CFA)followed by Incomplete Freund's adjuvant (IFA) once every other week fora total of three injections. Three days before fusion, the tail vein wasinjected with 50 ug EPRB-39 SAM IgG in 90 ul PBS. The titer wasestimated at 1/10000 from ELISA with high background

JH80 is the fusion number for EPRB-39. The following is a list of theclones and limiting dilution clones that are active:

7D8.29.19.47*, 39, 66 IgG2b k 8G9.3.4.51.22*, 30, 46 IgG2b k2A4.20.44.77*, 13, 14 IgG2b k

7D47, 8G9 and 2A77 indicate preferred subclones. The antibodies producedappear to be neoepitope specific because they don't react with a peptidethat spans the C-terminus cleavage site of SAA.

Example IV Antibody Binding to Aggregated and Soluble AA

Serum titers (determined by serial dilution) and monoclonal antibodybinding to aggregated AA were performed by ELISA as previously describedby Schenk D. et al., (1999) Nature 400, 173-177. Soluble AA refers tothe AA fibrils sonicated in dimethyl sulfoxide. Serial dilutions ofantibody were incubated with 50,000 cpm of ¹²⁵I-AA overnight at roomtemperature. 50 μl of a slurry containing 75 mg/ml protein A sepharose(Amersham Biosciences, Uppsala, Sweden)/200 μg rabbit anti-mouse IgG(H+L) (Jackson ImmunoResearch, West Grove, Pa., USA) was incubated withthe diluted antibodies for 1 hr at room temperature, washed twice, andcounted on a Wallac gamma counter (PerkinElmer Life Science, Grove,Ill., USA). All steps were performed in radioimmunoassay bufferconsisting of 10 mM Tris, 0.5 M NaCl, 1 mg/ml gelatin, and 0.5% NonidetP-40, pH 8.0.

Example V Analysis of Vλ6 Wil Structure

The sequences of the expressed human Vκ and Vλ immunoglobulin lightchain germline genes are as illustrated in FIGS. 21 and 22. Withexception of the κ1a, λ1a, λ3a, and λ3c subgroups there is a Glu-Aspresidue pairing at positions 81 and 82 in all, Vκ and Vλ germline genesequences (FIGS. 21 and 22). In addition, a second germline encodedGlu-Asp pairing at positions 50 and 51 is unique to Vλ6 germline gene.Thus, Vλ6 Wil contains both the 50-51 and 81-82 Glu-Asp pairs. The sidechains of residues 50 and 51 are both accessible on the surface of Vλ6Wil, as shown by x-ray crystallography (FIG. 24). In contrast, only theGlu81 side chain is surface exposed and the Asp82 side chain ispartially buried and appears to interact (either by electrostaticinteractions or H-bonding) with the side chains of Lys79 and Arg61 (FIG.25).

Based on these analyses of the x-ray crystal structure and the relativeavailability of the Glu-Asp side chains Applicants conclude that theburied Glu81 becomes accessible as the domain enters an aggregated(e.g., fibrillar) structure (or becomes partially denatured), thusexposing what is otherwise a hidden, cryptic epitope.

Example VI Analysis of Anti-AA Monoclonal Antibody Binding to Vλ6

A. Surface Plasmon Resonance

Surface plasmon resonance was used to establish the binding kinetics ofseveral monoclonal antibodies with Vλ6 Wil fibrils and monomer. At aconcentration of 6.6 nM all 3 antibodies bound to the immobilizedsynthetic Vλ6 Wil fibrils with a KD of ˜1 nM—a value comparable to thatfound for their reactivity with murine AA fibrils (FIG. 26). Thedeflection (expressed in RU) during the binding phase was similar formAbs 7D8 and 2A4 but was 50% lower for 8G9. This suggests that thedensity of this antibody on the fibrils was lower than the other 2reagents, as the calculated affinities were similar for all 3antibodies. An IgG1 mAb served as a control and exhibited no binding toVλ6 Wil fibrils.

Titration of the mAb 7D8 over the range of 6.6 nM to 33.3 nM producedthe expected decrease in the maximal deflection associated with kon(FIG. 27). In general, the binding kinetics were similar at eachconcentration, although in these pilot experiments the KD value for 7D8at 26.6 nM did differ from that obtained at the other concentrations.

To assess the specificity of the reaction and ensure that the binding ofthe mAbs with the fibrils occurred via the classic F(ab)-antigeninteraction (as opposed to Fc-mediated binding or non-specificadsorption), binding data were acquired in the presence of the immunogenpeptide (p39) at 20 and 1 μg/mL (FIG. 8). Peptide p41 which does notbind the mAb 7D8 at low concentrations, served as a control. In thepresence of 20 μg/mL p41 peptide, the binding kinetics for mAb 7D8 withVλ6 Wil fibrils were identical to 7D8 alone. In contrast, the immunogenpeptide p39 at 1 μg/mL caused a >2-fold decrease in the extent ofbinding as judged by the deflection of the measured signal (FIG. 28)Inhibition of fibril binding by 7D8 was almost completely inhibited when20 μg/mL of p39 peptide was used. These data indicated that mAb 7D8bound fibrils via the F(ab) region of the molecule inasmuch as thisinteraction could be completely inhibited by the immunogen peptide.

The reactivity of the mAb 7D8 with Vλ6 monomer immobilized on a chip wasexamined using the BIAcore. The antibody did not react with themonomeric protein. These data indicate that the binding site recognizedby the mAb 7D8 is present on fibrils, but not on the soluble precursorprotein, implying that the antigen is conformational or cryptic innature.

B. Immunohistochemistry

Immunohistochemistry was performed as follows: 6 μm-thick sections, cutfrom formalin-fixed, paraffin embedded blocks, were subjected to andantigen retrieval by incubation with CitraPlus (BioGenex, San Ramon,Calif.) for 30 min at 90° C. Tissues were immunostained with a 3 μg/mLsolution of mAbs 2A4, 7D8, or 8G9. The IgG2a mAb TY11 served as acontrol. A HRPO-conjugated horse anti-mouse Ig antibody (ImmPRESSUniversal Reagent, Vector Labs, Burlingame, Calif.) was used as thesecondary reagent. Slides were developed using 3,3′-diaminobezidene(Vector Labs) and examined using a Leica DM500 microscope. Theinteraction of the monoclonal antibodies with ALκ and ALλ amyloidtissues deposits was also studied using immunohistochemistry. Asillustrated in FIG. 29, amyloid deposits in a patient's thyroid glandwhich were composed of λ2 fragments were immunostained by 7D8, 2A4 and8G9. The areas of reactivity correlated with the amyloid deposits,indicated by the green-gold birefringence seen in the Congo red-stainedtissue section. The most impressive reactivity was achieved with mAbs7D8 and 2A4 mAbs while 8G9, although positive, was considerably weaker.These qualitative data correspond well with the BIAcore analyses inwhich 8G9 bound less to the Vλ6 Wil fibrils than the other 2 reagents(FIG. 26). The isotype matched mAb TY11 that served as a controlexhibited no amyloid immunoreactivity.

The amino acid sequence of this λ2 protein (SEQ ID NO: 86) (shown below)contains the germline encoded Glu and Asp residues at position 81 and82, respectively.

1         11         21         27d    35GSVVTQPPS VSGAPRQTVA ISCSGSSSNI GNNAVN WYQQLPGKAP45         55         65       73 KVLIYYDDLL PAGVSDRFSG SKSGTSAS LAIRGLGSED  83         93EGDYYCAAWD DSLSAL

Examination of an ALκ amyloid tissue deposit revealed 2A4, and to alesser degree the 7D8 and 8G9, to have positive reactivity. Again therewas concordance between the immunostaining and birefringent, congophilicamyloid regions. The TY11 mAb was unreactive.

C. Radioimaging of AL Amyloidoma Using ¹²⁵I-Labeled 7D8

The experimental in vivo model of AL amyloidoma was used to study ifradiolabeled mAb 7D8 would image human AL amyloid. The radiolabellingefficiency of 7D8, as determined by SDS-PAGE, revealed that both the IgHand IgL chains incorporated the 1-125 label, and no evidence of bandsassociated with fragmentation or aggregation were observed. SPECT/CTimaging of a mouse bearing an induced AL amyloidoma revealed that the¹²⁵I-labeled antibody localized to the induced, dorsally-located amyloidmass, as evidenced by accumulation of the radiolabeled antibodies in theamyloid, relative to amyloid-free tissues (e.g., liver, heart, spleen,and kidneys). Radioloabeled irrelevant IgG mAb did not accumulate in themass; however free radioiodide was observed accumulating in the thyroid,indicative of the catabolism and dehalogention of the IgG antibody. Thedistribution of the ¹²⁵I-7D8 mAb in the amyloidoma-bearing mice wasquantified by measuring the activity associated with the amyloid mass ascompared to that of the liver, spleen, kidney, stomach, heart, and lung.These data confirmed the SPETC/CT imaging study. At 72-h post injection(at which time the images were acquired and the tissues harvested), theamyloidoma contained ˜8% ID which is ˜4-fold higher than that seen inthe liver—the site of mAb catabolism—and the heart where residualblood-pool activity would expected to be high. The activity shown in thelung was due to the mode of euthanasia (data not shown).

To confirm the biodistribution data, the amyloidoma as well as theliver, spleen, heart, and kidneys were harvested and tissue sectionsprepared for autoradiographic analysis. Radiolabelling was performed asfollows: The 7D8 antibody was labeled with 2 mCi of reductant-free ¹²⁵I(Perkin Elmer) using limiting amounts of Chloramine T and suspended inPBS containing 5 mg/ml of bovine serum albumin (BSA/PBS). Unboundisotope and protein aggregates were removed by size-exclusion liquidchromatography through an Ultrogel AcA34 column (Amersham Pharmacia).Fractions containing IgG monomer were pooled for imaging experiments.The radiochemical yield was ˜50%, providing a specific activity of ˜25μCi/μg. ¹²⁵I-labeled mAb was subjected to SDS/PAGE (10% gels) in thepresence or absence of a reducing agent and analyzed with a Cyclonephosphor-imager. In accordance with the SPECT imaging andbiodistribution measurements, the autoradiographs confirmed significantaccumulation of ¹²⁵I-7D8 in the amyloidoma, relative to the liver. Therewas no evidence of uptake of radiolabeled antibody ¹²⁵I-7D8 in any otherorgans (other than the expected hepatic activity associate withcatabolism of the antibody). Although mAb 7D8 was relatively uniformlydistributed throughout the bulk of the amyloid mass, a moderately higherdensity was observed in the peripheral areas at the abdomen-amyloidboundary. There was no uptake of the radiolabeled control IgG in anyorgans.

D. Summary and Conclusions

Surface plasmon resonance, immunohistochemistry and in vivo radioimagingestablish that AA-reactive antibodies 2A4, 7D8, and 8G9 bind AL amyloidand fibrils (Kd˜I nM) derived from immunoglobulin light chains. Thisinteraction likely occurs at the highly-conserved Glu and Asp aminoacids at position 81 and 82, respectively, which form a cryptic linearepitope that becomes exposed only when the amyloidogenic light chain isincorporated into fibrils.

Example VII ELISA Analysis Demonstrates Antibody Binding to X₁EDX₂Peptides

BIAcore analysis was performed to evaluate binding of antibodies 2A4,7D8 and 8G4 on peptides of various sequences. As shown below in Table 4,the antibodies were found to react with peptides having the sequenceX₁EDX₂. Interestingly, the antibodies did not react with peptides havingadditional C-terminal residues. This suggests that the antibodiesspecifically bind to a neoepitope generated cleavage of SAA to generatea free C-terminal end However, as demonstrated in Example V, the freeend is not essential for binding of these antibodies to Vλ6 Wil, butrather the X₁EDX₂ domain adopts a conformation favorable to binding tothe antibodies as it enters an aggregated (e.g., fibrillar) structure(or becomes partially denatured), exposing an otherwise hidden, crypticepitope.

TABLE 4  Antibody Peptide pos/neg 2A4(39) CGGHEDT, (SEQ ID NO 87) POS 40CGGAEDS, (SEQ ID NO: 88) pos 41 GHEDTIADQE, (SEQ ID NO: 89) NEG 64CGGAEDT, (SEQ ID NO: 90) POS 65 CGGHADT, (SEQ ID NO: 91) WEAK 66CGGHEAT, (SEQ ID NO: 92) NEG 67 CGGHEDA, (SEQ ID NO: 93) POS 68CGGHEDTM, (SEQ ID NO: 94) NEG 69 CGGHEDTMA, (SEQ ID NO: 95) NEG 70CGGHEDTMAD, (SEQ ID NO: 96) NEG 71 CGGHED, (SEQ ID NO: 97) FALSE POS?7d8(39) CGGHEDT, (SEQ ID NO: 87) POS 40 CGGAEDS, (SEQ ID NO: 88) POS 41GHEDTIADQE, (SEQ ID NO: 89) NEG 64 CGGAEDT, (SEQ ID NO: 90) POS 65CGGHADT, (SEQ ID NO: 91) NEG 66 CGGHEAT, (SEQ ID NO: 92) NEG 67CGGHEDA, (SEQ ID NO: 93) POS 68 CGGHEDTM, (SEQ ID NO: 94) NEG 69CGGHEDTMA, (SEQ ID NO: 95) NEG 70 CGGHEDTMAD, (SEQ ID NO: 96) NEG 71CGGHED, (SEQ ID NO: 97) NEG 8g4(39) CGGHEDT, (SEQ ID NO: 87) POS 40CGGAEDS, (SEQ ID NO: 88) POS 41 GHEDTIADQE, (SEQ ID NO: 89) NEG 64CGGAEDT, (SEQ ID NO: 90) POS 65 CGGHADT, (SEQ ID NO: 91) NEG 66CGGHEAT, (SEQ ID NO: 92) NEG 67 CGGHEDA, (SEQ ID NO: 93) WEAK 68CGGHEDTM, (SEQ ID NO: 94) FALSE +? 69 CGGHEDTMA, (SEQ ID NO: 95 FALSE +?70 CGGHEDTMAD, (SEQ ID NO: 96) NEG 71 CGGHED, (SEQ ID NO: 97) NEG

Example VIII Immunohistochemical Analysis of Mouse AA

The reactivity of supernatants from hybridomas expressing antibodies2A4, 8G9 and 7D8 to murine AA splenic and hepatic amyloid deposits (theprincipal sites of amyloid deposition) was documentedimmunohistochemically. For these studies, sections of tissue harvestedfrom a TRIAD mouse with extensive AA amyloid in the liver and spleen (asevidenced by green birefringent Congophilic deposits) were stained withthe mAb-containing supernatants. All 3 bound to the hepatic and splenicamyloid. In contrast, there was no reactivity with culture supernatantsderived from irrelevant hybridomas. The capability of the amyloid using2A4, 8G9 and 7D8 to immunostain amyloid in fresh (unfixed), OCT-embeddedmurine liver and spleen was tested. There was evidence that the mAbsretained their ability to bind AA amyloid in the hepatic sinusoid. Inaddition, the antibody reactivity with splenic tissue was easier tointerpret, and the perifollicular amyloid was intensely immunostained.To demonstrate that the mAbs was specifically bound AA amyloid, the mAbsupernatants at a 1:25 dilution were preincubated with 50 μg/mL ofeither peptide #39 (p#39) or #41 (p#41) for 1 h at room temperature.With formalin-fixed tissue as a substrate, the p#39 peptide (50 μg/mL)significantly inhibited the amyloid reactivity of both 2A4 and 7D8 mAbs(the results with 8G9 are pending). In contrast, the p#41 peptide wasineffective. Comparable results were obtained with fresh tissues.

Example IX Immunohistochemical Analysis of Human AA

Comparison of the amino acid sequence of mouse and human SAA fromposition 73-76 reveals 2 identical residues, a conserved Ser to Thrsubstitution, and a non-conserved Ala to His exchange. To test if the2A4, 8G9 and 7D8 mAbs would cross-react with human AA amyloid deposits,we tested their reactivity to human AA-containing kidney, adrenal, ovaryand liver. In all cases, the mAb supernatants immunostained the amyloiddeposits. In ovarian tissue, the p#39 peptide effectively blocked thebinding of the mAbs to the perivascular AA amyloid, whereas the p#41peptide did not inhibit this reaction.

Example X Interaction of Anti-AA of Culture Supernatants withMurine-Derived AA Fibrils

The interaction of 2A4, 8G9 and 7D8 mAbs with AA amyloid was initiallytested by ELISA and the data, given in FIG. 31, analyzed using SigmaPlot(SPSS Inc.). Each point represents the mean±SE (n=3). A culturesupernatant from an irrelevant hybridoma was used as a control (CtrlCulture Sup). There was an extremely low signal-noise ratio and theresults showed that the first harvest contained more mAb relative to thesecond, as evidenced by the greater absorbance signal relative to thecontrol supernatant. (In addition, the immunohistochemical reactivity ofthe day 1 material was greater than the day 2 samples). Although the SEvalues were large, it appeared from these data that the binding affinityof 2A4, 8G9 and 7D8 was approximately equivalent with reactivity absentafter ˜1:64 dilution. The binding data also suggest that the capacity,i.e., the amount of mAb bound, varied with 7D8>8G9>2A4; however, thesedata were not corrected for mAb concentration and in subsequent studiesthis trend was not observed. Because of the low signal and highvariability found with the culture supernatants and to determine moreaccurately the relative binding affinity of the mAbs for murine andhuman AA amyloid fibrils (as well as to provide material for in vivobiodistribution studies) it was necessary to isolate the mAbs by proteinA affinity chromatography. The purity of the isolated mAbs wasestablished SDS-PAGE using 10% acrylamide gels under reducing andnon-reducing conditions (FIG. 32). Samples in lanes 1-4 treated withmercaptoethanol, lanes 5-9 without. Gel was stained with Coomassie blue:mAb 8G9, lanes 1 and 6; mAb 2A4, lanes 2 and 7; mAb 7D8, lanes 3 and 8;SP2/0 control supernatant, lanes 4 and 9; blank, lane 5. Protein Mrmarkers (Std) are, form top to bottom: 176, 119, 75, 49, 39, 25 and 19kDa. The interaction of the purified mAbs with immunizing peptide p#39,control peptide (p#41), murine and human AA extracts were determined byELISA as described above. These data were analyzed by fitting asigmoidal curve using the SigmaPlot software and the mAb concentrationat 50% saturation (EC50), determined (Table 5).

TABLE 5 EC₅₀ values for purified mAb binding Substrate mAb Human AAMouse AA (AEF) Peptide 39 Peptide 41 8G9 31.7 nM 5.64 nM 4.0 nM >>100 nM2A4 26.4 nM 4.09 nM 3.4 nM >>100 nM 7D8 13.3 nM 1.84 nM 2.3 nM >>100 nM

The interaction of the 3 mAbs with peptide p#39 exhibited saturablebinding with EC50 values in the low nanomolar range (see above Table 5).In contrast, even at the highest concentration of mAb used (100 nM)there was little detectable binding to the p#41 peptide (FIG. 33—Eachpoint represents the mean±SE, (n=3 at each concentration)). These dataconfirmed the immunohistochemical results described above, i.e., thatpeptide p#39 was capable of completely blocking the binding of the mAbsto AA amyloid laden tissues. The calculated EC50s for the binding ofeach mAb with p#39 peptide were essentially identical as was the casewhen a murine AA amyloid extract was used as the substrate (FIG. 34—Eachpoint represents the mean±SE (n=3 at each concentration)). Thecalculated EC50 values for the mAbs binding to mouse AA extract wereessentially identical to those obtained when the p#39 peptide was usedas the substrate (FIG. 34; Table 5). In contrast, when human AA amyloidextract was dried onto the wells of the microplate, the EC50 values werebetween 5 and 7× lower than that observed for mouse AA and peptide p#39(FIG. 35—Each point represents the mean±SE (n=3 at each concentration);Table 5). Because the EC50 value for 7D8 mAb binding was the lowest ofthe 3 antibodies tested, Applicants selected this reagent for in vivoco-localization and imaging studies. The 2 amino acid substitutions inthe human SAA sequence with respect to the murine protein affected theEC50 values. While not wishing to be bound by a particular theory,Applicants attribute the higher EC50 for the human AA to a poorer “fit”of the amino acid side chains in the antigen binding site. however, thiseffect corresponds to only a 5-fold decrease in the relative affinitywhen the amyloid extracts are surface adsorbed, as in the ELISA.Furthermore, these data support the observation that all 3 mAbs bound toboth murine and human tissue AA amyloid deposits.

Example XI Competitive Binding of Mabs to Mouse and Human AA Amyloid

To determine the effect, if any, of potential denaturation when adsorbedto the surface of the microtiter well, the reactivity of the 2A4, 8G9and 7D4 was evaluated using a competition ELISA in which murine or humanAA amyloid extract was used as a soluble competitor for the interactionof the mAbs with surface-bound AA extract.

In all cases, soluble (non-adsorbed) AA amyloid fibrils of both humanand mouse origin were capable of competing for the 3 mAbs, indicatingthat the epitope recognized by the reagents is not dependent upon thepartial denaturation that results from surface adsorption. In general,the murine AA (AEF) extract was a better competitor than the human AA(Table 6).

TABLE 6 IC₅₀ values (μg/mL) for mAb binding to AA amyloid mAb HumanAA^(†) Mouse AA (AEF)^(‡) 8G9 >119.5 17.3 2A4 >211.7 14.7 7D8 >881.126.8 ^(†)Human AA amyloid in solution competing for adsorbed mouse AA(AEF); ^(‡)Mouse AA (AEF) in solution competing for adsorbed human AAamyloid extract on plate.

The IC50 values (concentration of AA (by weight) that reduced the mAbbinding by 50%) for murine AEF in solution were ˜20 μg/mL, whereas forhuman AA the values were 6- to 44-fold greater (in contrast, the EC50sfor human AA were only 7-fold lower than those for mouse AA). This mayreflect the fact that, when in solution, the epitope on the amyloidfibrils is less accessible in human AA preparations as compared tomurine AA.

As expected, the 7D8 mAb that exhibited the highest relative affinityfor the human and murine AA fibrils when they were surface-adsorbedrequired the highest concentration of AA amyloid to achieve competition.

Example XII Radiolabeled MAb 7D8

The radiolabeling efficiency of 7D8 was determined by SDS-PAGE. Reducedand native mAb were analyzed and the proteins visualized using aphosphor imager. Both the IgH and IgL chains incorporated the I-125label, and no evidence of bands associated with fragmentation oraggregation were observed.

Example XIII Imaging of AA Amyloid using ¹²⁵I-Labeled 7D8

To study the in vivo localization of radiolabeled mAb 7D8 three groupsof mice were used: transgenic IL-6; AgNO3/AEF induced, andamyloid-lacking controls (WT). The SPECT/CT imaging revealed that the¹²⁵I-7D8 mAb localized to murine AA amyloid deposits in the spleen andliver, as evidenced by the accumulation of the radiolabeled mAb in thesetissues relative to the control mouse, which showed only low blood poolactivity in the liver and free iodide the thyroid gland.

In contrast to these mice, the AgNO3-injected mouse showed thyroiduptake of free iodide, some hepatic activity, but the major site of¹²⁵I-7D8 binding was seen at the site of s.c. AgNO3 injection (the lowerright dorsal area). The activity in this area is clearly circumscribedby the x-ray-attenuating silver solution as seen by CT. The 7D8 mAb hasbeen shown to bind to AA amyloid deposits in both the liver and spleenin the presence of circulating sAA in the TRIAD mouse, as evidenced inthe SPECT images.

A. Biodistribution of ¹²⁵I-7D8 in Mice.

48 h post-injection of ¹²⁵I-7D8 there was radioactivity in the bloodpool, which accounted for the relatively high uptake In the lung (whichfill with blood when the mice are sacrificed). Of note, thehepatosplenic accumulation of mAb in the IL-6 mouse is indicative of thepresence of amyloid. The SPECT/CT images confirmed the distribution ofthe mAb in these organs. 72 h post-injection the blood pool values havechanged little as evidenced by the unchanged activity in the heart andlung relative to the mice sacrificed at 48 h, due to the relatively longT_(1/2bio) for this mAb (˜60 h). There was significant accumulation ofthe radiolabeled mAb in the IL-6 mouse, which correlated with the SPECTimages that were acquired showing impressive splenic and, to a lesserdegree, hepatic uptake. Of the other organs, most important was theliver (which is the site of catabolism of IgG and the source of sAAduring the acute phase response). In the WT mice, with no inflammatorychallenge or amyloid, the liver contained <6% ID/g, which is comparableto the kidney and heart where the blood pool contributes almostexclusively to the signal.

B. Autoradiographic and Histochemical Analyses.

In order to determine if the increased hepatic accumulation of ¹²⁵I-7D8in the IL-6 and AgNO₃ mice resulted from amyloid uptake, catabolicclearance or binding to newly synthesized sAA, liver as well as othertissues were subjected to autoradiographic analysis.

Based on the SPECT imaging and biodistribution measurements, it waspresumed that the greatest amount of amyloid in the transgenic IL-6 micewas in the liver and spleen. This supposition was confirmed in the Congored-stained sections in which significant amyloid was observedthroughout the red pulp as well as in the perivascular regions andsinusoids of the liver. Additional, more discreet birefringent depositswere present in the kidneys and heart. The distribution of the ¹²⁵I-7D8within these tissues correlated well with the Congo red and AA-reactivematerial. There was no accumulation in hepatocytes that were devoid ofamyloid.

Based on the biodistribution data, the AgNO₃-treated mouse had moreuptake of 125I-7D8 in the liver than the spleen, which was unexpectedsince this is not the normal pattern of accumulation of AA in suchanimals. Congo red-staining revealed small amounts of amyloid in asingle perifollicular region in the spleen (upper right corner) andextensive hepatic perivascular deposits both of which were evident inthe autoradiographs. Additionally, the s.c. site of the AgNO₃ injectionwas seen in the SPECT images to have a significant concentration of¹²⁵I-7D8 (we also have observed this when radioiodinated SAP was used asthe imaging agent). This site does not contain amyloid (i.e., Congored-birefringent material); however, it was immunostained by anti-AAmAb. Without wishing to be bound to a particular theory, it is possiblethat the mAb 7D8 localizes to sites of inflammation or “pre-amyloid” (aswell as mature amyloid deposits). In contrast to the impressiveaccumulation of 7D8 in the organs of the IL-6 mouse, the tissues of thecontrol mice were found to have little or no tracer in any organ otherthan the blood pool. No amyloid was found in Congo red-stained sectionsof any organ of these controls.

C. Pharmacokinetics of ¹²⁵I-7D8.

After injection of the radiolabeled 7D8 antibody, the rate ofdisappearance of the molecule was determined and the half-lifedeterminations summarized in Table 7. These results indicated that theT_(1/2bio) of 7D8 was ˜60 h, consistent with that of an IgG2b murine mAb(note, 7D8 is of the IgG2b subclass). The slightly more rapid clearanceof the ¹²⁵I-7D8 in the IL-6 (TRIAD) mice was not considered significant.Based on these data, retention of the mAb by tissue amyloid, asevidenced in the SPECT data, over 72 h does not influence the excretionrate.

TABLE 7 half-life analyses for ¹²⁵I-7D8 in mice Mouse A (S.E.) K (S.E. ×10⁻⁴) R² t_(1/2 bio) t_(1/2 eff) IL-6, 48 h 191.7 (2.96) 0.0117 (7.0)0.98 59.2 h 56.2 IL-6, 72 h 175.2 (3.99)  0.012 (8.9) 0.97 57.7 h AgNO₃,48 h 181.0 (1.99) 0.0106 (4.9) 0.99 65.3 h 61.1 AgNO₃, 72 h 174.1 (2.97)0.0112 (5.8) 0.98 62.2 h Ctrl, 48 h 185.1 (3.19) 0.0108 (7.6) 0.98 64.3h 61.3 Ctrl, 72 h 185.1 (3.09) 0.0109 (5.6) 0.98 63.7 h

1. Method of Identifying Agents that Prevent or Treat Amyloidosis UsingTransgenic or TRIAD Mouse. Procedures for preparation of agents aredescribed in Schenk et al. Nature 400:173-177. Agents are emulsified 1:1(v/v) with complete Freund's adjuvant for the first immunization oftransgenic mice, followed by a boost in complete Freund's adjuvant at 2weeks and monthly thereafter. PBS injections followed the same scheduleand mice were injected with 1:1 mix of PBS/adjuvant for control. Thelife span of the transgenic mice is compared to determine whether theagents are effective in preventing AA Amyloidosis by increasing the lifeof the animal.

2. Histopathology. For light and polarizing microscopy, 4- to 6-μm-thicktissue sections were cut and stained with hematoxylin and eosin (HE) anda freshly prepared alkaline Congo red solution, respectively. Forelectron microscopy, sections were embedded in Epon (Ted Pella, Redding,Calif.), sectioned, and examined with a JEOL 100S transmission electronmicroscope. See Ludlage et al. Vet Pathol 42:117-124 (2005).

3. Immunohistochemistry. Paraffin-embedded tissue sections (6-μm-thick)were cut on a microtome, mounted on poly-L-lysine-coated slides, driedovernight at room temperature, and deparaffinized. Immunostaining wasperformed using the avidinbiotin complex (ABC-elite) technique asdescribed previously. The primary antibodies were mouse anti-humanamyloid A (Accurate Chemical and Scientific Corporation, Westbury, N.Y.)and anti-mouse SAA polyclonal antisera. Affinity-purified horseanti-mouse immunoglobulin-G (IgG) horseradish peroxidase conjugate(Vector Laboratories, Burlingame, Calif.) or goat anti-rabbit, -mouse,or -rat IgG horse-radish peroxidase conjugates (BioRad Laboratories,Richmond, Calif.) were used as the secondary antibodies.

4. SAA Quantitation by ELISA. SAA concentrations were measured by anenzyme-linked immunosorbent assay (ELISA) using the Multispecies SAAELISA kit according to directions supplied by the manufacturer(Biosource, Camarillo, Calif.). Standard curves were prepared usingknown amounts of human SAA protein and absorbance was measured at 405 nmwith a model 4450 BioRad plate reader (Fullerton, Calif.).

5. Radiolabeled SAP Scintigraphy Turnover Studies in Mice. SAP wasoxidatively iodinated with ¹²⁵I (2-5 MBq/mg) by usingN-bromosuccinimide. 6-12 weeks old mice received 2-10 μg of ¹²⁵I-SAP in200 μL intravenously. Precisely measured tail bleeds (0.01-0.04 g) weretaken at specific time intervals and trichloroacetic acid-precipitableradioactivity was counted in the same run at the end of each experimenttogether with standard aliquots of the injected tracer. Pepys et al.Proc Natl. Acad. Sci. USA 91:5602-5606 (1994).

6. Radiolabeled SAP Scintigraphy Turnover and Imaging Studies in Man.SAP for use in man was isolated from the plasma of a single normalaccredited donor and was oxidatively iodinated with ¹²⁵I (2-5 MBq/mg) or¹²³I (110 MBq/50 μg of protein) by using N-bromosuccinimide. Afterinjection of ¹²³I SAP, data were acquired and processed on an IGEStarcam gamma camera (IGE Medical Systems, Slough, U.K. Clearance of¹²⁵I-labeled SAP was studied in healthy individuals and patientssuffering from AA amyloidosis. Pepys et al. Proc Natl. Acad. Sci. USA91:5602-5606 (1994)

7. Amyloid Extraction and Purification. The methods used to extractamyloid from tissue were as described by Pras et al. See Pras et al. J.Clin. Invest. 47:924-933 (1968) In brief, a portion of liver or tissuesfrom other organs obtained at necropsy and maintained at −80 C washomogenized with cold saline in an ice bath using an Omni-Mixer (OmniInternational, Waterbury, Conn.). The extract was centrifuged at 10,000rpm for 30 minutes at 4 C and the pellet reextracted twice more withcold saline, once with 0.1 M sodium citrate Tris-buffered saline, pH8.0, and then again with saline until the A280 of the supernatant was<0.10. The resultant pellet was homogenized with cold distilled water,and the mixture centrifuged at 35,000 rpm for 3 hours at 4 C. The pelletobtained from the water extract was then lyophilized.

8. Surface Plasmon Resonance. Binding kinetics were measured on aBIAcore X instrument. Fibrils prepared from the V_(λ)6 Wil weresonicated briefly with a probe sonicator and then coupled to a CM-5 chipusing amine chemistry, as per the BIAcore protocol. This processutilizes EDC and NHS to activate the carboxyl groups on the chip forcoupling with free amino groups on the fibrils. Coupling was conductedin a NaOAc buffer, pH 4.0 at a concentration of 100 μg/mL. The controlchannel was “mockcoupled” and both channels were reacted withethanolamine to saturate unreacted sites. Approximately 16,000 RU ofV_(λ)6 Wil fibrils were coupled.

Sensograms were run in HBS-EP buffer from BIAcore at 20 μL/min in theFc1 (V_(λ)6 Wil fibrils) minus Fc-2 (control) mode. Samples containingmAb or mAb plus peptide inhibitors were injected (70 μL) and thesensograms collected using the delayed-wash function for 200 sec. Datawere analyzed in the BIAevalutation software, using the 1:1 Langmuirmodel with mass-action correction.

9. MicroSPECT/CT. Two cohorts of 3 mice each were injected s.c. with 50mg of human AL amyloid extract between the scapulae. After 7 days, onegroup of mice received an iv tail vein injection of ˜300 μCi of¹²⁵I-labeled mAb 7D8. The second group were administered and equalquantity of murine mAb MOPC 31 C as a control. After 72 hr, the micewere sacrificed by isoflurane overdose and SPECT/CT images acquired. Toprovide vascular contrast-enhancement in the CT images, mice were givena 200-μL iv dose of Fenestra VC™ (Advanced Research Technologies,Montreal, Canada) 5 min prior to scanning.

SPECT data were collected with a microCAT II+SPECT dual modality imagingplatform (Siemens Preclinical Solutions, Knoxyille, Tenn.), capable ofsubmillimeter spatial resolution when equipped with a 0.5 mm-porediameter pinhole collimator. When imaging, the 2 detectors (composed ofa 50 mm-diameter Hamamatsu R2486-02 multi-anode photo-multiplier tubecoupled to a 1×1×8 mm CsI (Tl) crystal array arranged on a 1.2 mm² grid)were positioned ˜45 mm from the center of rotation. Each SPECT datasetcomprised 45 projections collected over 360° during the course of ˜50min. Images were reconstructed using an implementation of theexpectation maximization-maximum likelihood (EM-ML) algorithm.

After collection of SPECT data, high-resolution CT images were obtained.The microCAT II scanner has a circular orbit cone beam geometry,equipped with a 20-80 kVp microfocus x-ray source, and captures a 90mm×60 mm field of view using a 2048×3072 CCD array detector, opticallycoupled to a minR phosphor screen via a fiber-optic bundle. Each CTdataset, composed of 360 projections at 1° azimuths, was acquired in 8min. Images were reconstructed in real-time on isotropic 77-μm voxelsusing an implementation of the Feldkamp backprojection algorithm.

To facilitate co-registration of the reconstructed SPECT and CT images,Co-57 sealed sources were placed on the imaging bed. The microSPECT andCT datasets were visualized and co-registered manually with a 3-D imageanalysis software package (Amira, Version 3.1: Mercury ComputerSystems).

10. Biodistribution. Samples of liver, spleen, kidney, heart, lung, andimplanted amyloid tumors (i.e., amyloidoma) were harvested from the miceand placed into tared vials, weighed, and the radioactivity measured.The primary index values were expressed as % injected dose/g tissue (%ID/g).

11. Autoradiography. 6 μm-thick sections cut from formalin-fixed,paraffin-embedded blocks of tissue obtained from mice sacrificed 72 hpost-injection of ¹²⁵I-7D8 were placed on Probond microscope slides(Fisher Scientific), dipped in NTB-2 emulsion (Eastman Kodak), stored inthe dark, and developed after a 24-h exposure. The sections werecounter-stained with hematoxylin and eosin (H&E), cover-slipped usingPermount (Fisher Scientific), and examined by light microscopy. Inaddition, consecutive slides were stained with alkaline Congo red andviewed under cross-polarized illumination. Finally, a third slide wasimmunostained using as primary reagent our AA-reactive mAb. Digitalcamera microscopic images were taken and evaluated using an imageanalysis software package (Image Pro Plus, Media, Cybernetics).

Example XIV Preparation of Humanized 2A4 and 7D8 Antibodies

Humanized 2A4, 7D8, and 8G9 antibodies were prepared by grafting ofmurine 2A4, 7D8, and 8G9 CDRs onto human acceptor frameworks accordingto techniques known in the art. Back mutations were made to reduceantigenicity while preserving binding affinity. The light chain andheavy chain variable regions of murine 2A4 are set forth as residues20-131 of SEQ ID NO: 152 and as residues 20-138 of SEQ ID NO: 154,respectively. The light chain and heavy chain variable regions of 7D8are set forth as residues 20-131 of SEQ ID NO: 153 and as residues20-138 of SEQ ID NO: 154, respectively. The light chain variable regionsof murine 2A4 and 8G9 are identical to each other and differ from thelight chain variable region of 7D8 in a single residue in CDR1. Theheavy chain variable regions of each of 2A4, 7D8, and 8G9 are identical.

The variable kappa (Vk) of 2A4 and 7D8 belong to mouse subgroup 2, whichcorresponds to human subgroup 2 and the variable heavy (Vh) to mousesubgroup 3c which corresponds to human subgroup 3 (Kabat et al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition. NIHPublication No. 91-3242). CDR-L1 includes 16 residues and belongs tocanonical class 4 in Vk. CDR-L2 includes 7 residues and belongs to class1 in Vk. CDR-L3 includes 9 residues and belongs to class 1 in Vk. SeeMartin A C, Thornton J M. (1996) J Mol Biol. 263, 800-15. The leucine atposition 27 in the 7D8 is rather unusual, and the glutamine in 2A4 ismore usual. A model shows the sidechain is on the surface of the bindingsite, and therefore should be important for antigen binding. CDR-H1includes 5 residues and belongs to class 1, and CDR-H2 includes 19residues and belongs to class 4 (Martin & Thornton, 1996). CDR-H3 has nocanonical classes, but the 8 residue loop probably has a kinked baseaccording to the rules of Shirai et al. (1999) FEBS Lett. 455, 188-97.This is conserved in a model although the conformation of the apex ofCDR-H3 may be different. The residues at the interface between the Vkand Vh domains are the ones commonly found for 2A4 Vk, 7D8 Vk and 2A4Vh.

A search was made of the PDB database (Deshpande et al. (2005) NucleicAcids Res. 33: D233-7) to find structures which would guide the choiceof back mutations. A search of the non-redundant protein sequencedatabase from NCBI allowed selection of suitable human frameworks intowhich to graft the murine CDRs. For Vk, a human kappa light chain withNCBI accession code BAC01562 (gi:21669075) (SEQ ID NO: 166) was chosen.This has the same length CDR-L3 and belongs to human germline VKIIA19/A3and human kappa subgroup 2. A similar framework which only differed inthe J-region was also found with NCBI accession code BAC01733(gi:21669417) (SEQ ID NO: 167). BAC01562 was used as a framework for 2A4Vk, and BAC01733 was used as a framework for 7D8 Vk. For Vh, human Igheavy chain AAC51024 (gi:1791061) (SEQ ID NO: 165) was used. See Glas etal. (1997) Clin. Exp. Immunol. 107: 372-380. This belongs to humangermline VH3-72 and human heavy subgroup 3.

Representative humanized 2A4 light chain variable regions are set forthas SEQ ID NOs: 155, 156, and 157. Representative humanized 7D8 lightchain variable regions are set forth as SEQ ID NOs: 158, 159, 160, 174,175, and 176. Representative humanized 2A4/7D8 heavy chain variableregions are set forth as SEQ ID NOs: 161, 162, and 163. See FIGS.36A-36E.

Representative humanized antibodies of the invention include antibodieshaving a light chain variable region selected from one of residues20-131 of SEQ ID NO: 152, residues 20-131 of SEQ ID NO: 153, and SEQ IDNOs: 155, 156, 157, 157, 159, 160, 174, 175, and 176; and a heavy chainvariable region selected from one of residues 20-138 of SEQ ID NO: 154and SEQ ID NOs: 161, 162, and 163.

Example XV Therapeutic Effects of MAb 2A4 in Mice with Severe SystemicAA Amyloidosis

The therapeutic efficacy of mAb 2A4 was evaluated in H2/huIL-6 mice withsevere systemic amyloidosis. The transgenic H2/huIL-6 mice, whichconstitutively express a human IL-6 transgene, are prove to rapid andirreversible systemic AA amyloidosis. In a first and second study, micetreated with isotype-matched mAb TY-11, which has no reported activityin mice, was used as a control. Before administering the amyloidenhancing factor to induce AA, H2/huIL-6 mice were sampled and bled viathe retro-orbital sinus, serum prepared, and the sAA concentrationdetermined using a commercially available ELISA kit. Representativevalues were as follows: 2196.7 μg/mL, 823.91 μg/mL, 1415.00 μg/mL,1673.01 μg/mL, 814.53 μg/mL, 1088.18 μg/mL, 736.34 μg/mL, 1546.35 μg/mL,953.70 μg/mL, 886.46 μg/mL, mean=1213.4±478 μg/mL.

At the start of the second study (week 0), H2/huIL-6 mice were injectediv with 100 μg of amyloid enhancing factor (AEF). After induction of AApathology by injecting AEF, the mice were administered 5 injections of100 μg subcutaneously in alternate limbs of mAb 2A4(13 animals) or TY11(11 animals). The therapy was initiated at approximately 1 week post AEFinjection. The survival of animals in each treatment group was plottedand analyzed. The results are shown in Table 8. Only 45% of the mAbTY11-treated mice survived to the end of the study. In contrast, none ofthe 2A4-treated mice were lost over the course of the study. Analysis ofthe survival data using standard methods showed a significant differencein the survival curves (P<0.0025) in both groups. The median survival ofthe TY11-treated mice was calculated to be 41 days, comparable to thatobserved in a prior study (38.5 days).

TABLE 8 Percentage of animals surviving Days post injection TY11-treated2A4-treated 0 100.00 100.00 22 81.82 100.00 33 72.73 100.00 37 63.64100.00 41 45.45 100.00 42 45.45 100.00

At week 6, post-AEF, mice were bled and sacrificed, and their organsharvested for further analysis. For quantification of amyloid in liverand spleen, Congo red birefringence was visualized microscopically undercross-polarized illumination and digitally recorded. The area ofbirefringent material was determined by selecting (using a spectralsegmentation method) and quantifying the amyloid-associated pixels. Theamyloid burden index (ABI), a measure of amyloid content, was expressedas the percentage area occupied by amyloid in each organ. Quantificationof amyloid in the livers and spleens of 2A4 and TY11-treated micerevealed no significant difference between the two treatments. However,the TY11-treated mice that survived to day 42 for comparison with2A4-treated mice were those that did not develop a morbid degree ordistribution of AA amyloid to thereby result in morbidity. Thehepatosplenic amyloid burden is also monitored during the course of thesurvival study to assess an increase in amyloid burden that correlateswith morbidity.

In a third study, mAb 2A4 was compared to the isotype-matched mAb JH70,which has no reported reactivity in mice. In addition blood chemistryand other parameters were monitored throughout the treatment period.Male and female H2/huIL-6 mice born between Aug. 1, 2008 and Sep. 7,2008 were used in this study. Twenty three female mice and 16 male micewere bled via the retro-orbital sinus. Whole blood was used for chemicalcharacterization of blood urea nitrogen (BUN) and alanineaminotransferase (ALT) to measure renal and hepatic function by usingthe VetScan VS2 (Abaxis, Union City, Calif.). The serum concentration of12 other proteins and analytes were simultaneously measured. A completeblood count (CBC) was performed using the VetScan HM5 platform. Inaddition, each mouse was administered a low dose (˜50-60 μCi) ofradioiodinated human serum amyloid P component (¹²⁵I-SAP) in 5 mg/mLbovine serum albumin to assess the amyloid burden of the mice prior toinitiation of the disease process. The percent of ¹²⁵I-SAP retained at24 h post-injection (pi) was measured by placing each mouse into a dosecalibrator. Retention of ¹²⁵I-SAP greater than that observed innon-transgenic (control) mice was indicative of amyloid disease.Finally, serum was used to measure the concentration of serum amyloidprotein A (sAA) using a commercial ELISA assay. A summary of thesepretreatment data, selected blood chemistry values, and the treatmentsgiven to each mouse are shown below in Tables 9 and 10.

TABLE 9 Summary Of Pre-Treatment Data And MAb Therapy For Each Animal¹²⁵I-SAP sAA conc. retention Therapy Mouse # (μg/mL) Sex DOB (%) (GroupNo.) 3488 360 F Aug. 1, 2008 9 2A4 (1) 3489 996 F Aug. 1, 2008 29 2A4(1) 3490 472 F Aug. 1, 2008 10 2A4 (1) 3492 2068 M Aug. 1, 2008 13 2A4(1) 3493 1740 M Aug. 1, 2008 11 JH70 (1) 3494 1272 M Aug. 1, 2008 10JH70 (1) 3495 1436 M Aug. 1, 2008 13 JH70 (1) 3496 2080 M Aug. 1, 2008 92A4 (1) 3498 268 M Aug. 1, 2008 9 2A4 (1) 3500 700 F Aug. 11, 2008 11JH70 (1) 3501 ND F Aug. 11, 2008 9 JH70 (1) 3503 1040 F Aug. 11, 2008 11JH70 (1) 3504 960 F Aug. 11, 2008 10 JH70 (1) 3513¹ 4400 M Aug. 13, 200860 2A4 (1) 3514¹ 4400 M Aug. 13, 2008 40 2A4 (1) 3515 2800 M Aug. 13,2008 13 2A4 (1) 3521 1480 M Aug. 18, 2008 11 2A4 (1) 3524 1680 M Aug.18, 2008 9 2A4 (1) 3549 720 F Sep. 6, 2008 9 2A4 (2) 3550 760 F Sep. 6,2008 9 2A4 (2) 3552² 0 F Sep. 6, 2008 11 2A4 (2) 3553 1160 F Sep. 6,2008 12 2A4 (2) 3558 1660 M Sep. 6, 2008 9 JH70 (2) 3559 3520 M Sep. 6,2008 12 JH70 (2) 3562 1312 F Sep. 6, 2008 11 JH70 (2) 3563 1120 M Sep.6, 2008 9 JH70 (2) 3564 2512 M Sep. 6, 2008 11 2A4 (2) 3565 1960 M Sep.6, 2008 10 2A4 (2) 3567 1880 F Sep. 6, 2008 12 2A4 (2) 3570 792 F Sep.7, 2008 13 2A4 (2) 3573 700 F Sep. 7, 2008 8 2A4 (2) 3577² 0 F Sep. 7,2008 10 2A4 (2) 3578² 0 F Sep. 7, 2008 9 2A4 (2) 3579 1120 F Sep. 7,2008 10 2A4 (2) 3580² 0 F Sep. 7, 2008 8 JH70 (2) 3581 700 F Sep. 7,2008 9 JH70 (2) 3582 1680 F Sep. 7, 2008 9 JH70 (2) 3583 804 F Sep. 7,2008 9 JH70 (2) 3584 1040 F Sep. 7, 2008 14 JH70 (2) ¹homozygous IL-6animals with high sAA levels and amyloid disease early in life. ²wildtype mice without circulating sAA and no amyloid disease. ¹²⁵I-SAPretention in these animals is considered normal and reflecting noamyloid burden.

TABLE 10 Normal Values For Blood Chemistry Parameters In H2/huIL-6 MiceBUN GLU ALT ALB TP GLOB (mg/dL) (mg/dL) (U/L) (g/dL) (g/dL) (g/dL) F M FM F M F M F M F M Mean 21.1 23.8 144.7 151.2 37.6 42.3 2.5 1.9 5.6 6.23.1 4.4 SD 4.0 2.7 14.0 17.6 16.3 24.3 0.3 0.4 0.2 0.6 0.4 0.6 n 18 1318 13 18 13 18 13 18 13 18 13 High 28.0 30.0 184.0 179.0 79.0 105.0 3.02.6 6.0 7.4 3.7 5.8 Low 15.0 20.0 126.0 119.0 21.0 23.0 2.0 1.2 5.1 5.52.6 3.4 Median 20.0 24.0 143.0 154.0 32.5 32.0 2.4 1.9 5.6 6.0 3.2 4.3BUN, blood urea nitrogen; GLU, glucose; ALT, alanine aminotransferase;ALB, albumin; TP, total serum protein; GLOB, immunoglobulin; F, female;M, male; SD, standard deviation; n is the number of mice used todetermine the values.

At the start of the third study (week 0), all of the all the H2/huIL-6mice received 100 μg iv of amyloid enhancing factor (1 mg/mL). One weekthereafter, therapy began and each mouse was administered 100 μg ofeither mAb 2A4 or JH70 sc as outlined in Table 9. The mAb injectionscontinued weekly for 7 weeks.

At 2 wk post-AEF, CBC, blood chemistry, and serum sAA measurements weremade using blood collected via the retro-orbital sinus. At this timealso, the mice in group 1 were administered ˜60 μCi of ¹²⁵I-SAP in BSAas before, to assess the accumulation of amyloid as evidenced by theretention of the radiolabeled SAP. Several of the animals showed anadverse effect of extreme distress, and therefore, evaluation of amyloidburden using ¹²⁵I-SAP was discontinued. Results of selected bloodchemistry parameters, acquired 2 wk post-AEF are shown in Table 11.

TABLE 11 BUN GLU ALT ALB GLOB (mg/dL) (mg/dL) (U/L) (g/dL) TP (g/dL)(g/dL) F M F M F M F M F M F M Mean 31.4 52.1 145.1 129.8 33.9 63.3 2.31.8 6.5 8.1 4.2 6.2 SD 24.3 39.1 16.6 25.6 6.9 30.6 0.3 0.5 1.0 1.7 1.11.5 n 15 13 15 13 15 13 15 13 15 13 15 12 High 100.0 159.0 177.0 178.046.0 134.0 2.7 3.0 8.6 11.7 7.0 9.6 Low 16.0 20.0 104.0 82.0 22.0 32.01.7 1.0 5.2 6.0 3.1 4.5 Median 22.0 31.0 150.0 120.0 32.0 54.0 2.3 1.76.5 7.5 4.0 6.0 BUN, blood urea nitrogen; GLU, glucose; ALT, alanineaminotransferase; ALB, albumin; TP, total serum protein; GLOB,immunoglobulin; F, female; M, male; SD, standard deviation; n is thenumber of mice used to determine the values.

At 8 weeks post-AEF, the mice were bled a final time and immediatelythereafter were administered ˜200 μCi of ¹²⁵I-SAP using 5% normal mouseserum as carrier. In response to this treatment, a few animals showedsome unusual behavior that abated within 30 min. Twenty four hourslater, the mice were injected with x-ray CT contrast agent (˜200 μL ivin the tail vein) and were then sacrificed by isoflurane overdose.Single photon emission (SPECT) and x-ray (CT) tomographic images of eachanimal were acquired. The organs were harvested and the amount ofradioactivity in each sample was calculated and expressed as % injecteddose per gram of tissue. Additionally, a portion of each tissue wasfixed overnight in buffered formalin in preparation for sectioning andmicroscopic analysis.

During the 7 wk therapy study, 2 mice were found dead and 3 mice weresacrificed because they were deemed unlikely to survive overnight andhad a poor body condition score (<2; associated with >15% weight loss).Mice that experienced an adverse reaction to ¹²⁵I-SAP injection and 1mouse that was sacrificed due to complications that arose from aretro-orbital bleed were not evaluated as part of the survival analysis.The survival of the mice in each mAb treatment group is shown in Table12.

TABLE 12 Percentage of animals surviving Days post injectionTY11-treated 2A4-treated 0 100.00 100.00 41 100.00 42 100.00 53 85.71100.00 55 71.43 100.00 56 64.29 100.00 57 64.29 100.00

Approximately 65% of the mAb JH70-treated mice that were assessablesurvived to the end of the study. In contrast, none of the 2A4 mice thatwere assessable died during the 57 days. Analysis of the survival datausing the standard methods demonstrated a significant difference in thesurvival curves (P=0.015 using Mantel-Cox test and P=0.016 usingGrehan-Breslow-Wilcoxon test).

The final blood chemistry data were analyzed according to the therapythat each mouse received. Because of differences in the mean parametervalues associated with male and female H2/huIL-6 mice (at the time ofsacrifice, BUN levels in female mice were higher for both 2A4-treatedand JH70-treated mice), only the female mice that survived are includedin Table 13 below.

TABLE 13 BUN GLU ALT ALB GLOB (mg/dL) (mg/dL) (U/L) (g/dL) TP (g/dL)(g/dL) 2A4 JH70 2A4 JH70 2A4 JH70 2A4 JH70 2A4 JH70 2A4 JH70 Mean 60.773.3 107.8 100.1 45.5 119.7 2.3 2.2 9.2 9.1 7.0 7.1 SD 27.2 25.7 27.013.3 6.2 123.1 0.5 0.6 1.5 1.5 2.0 2.1 n 6.0 7.0 6.0 7.0 6.0 7.0 6.0 7.06.0 7.0 6.0 7.0 High 95.0 120.0 160.0 123.0 52.0 381.0 2.9 3.0 11.7 11.910.1 10.6 Low 17.0 36.0 83.0 83.0 35.0 33.0 1.5 1.2 7.2 7.5 4.3 5.3Median 66.5 70.0 99.5 98.0 46.5 65.0 2.2 2.1 9.1 8.9 7.1 6.2 BUN, bloodurea nitrogen; GLU, glucose; ALT, alanine aminotransferase; ALB,albumin; TP, total serum protein; GLOB, immunoglobulin; F, female; M,male; SD, standard deviation; n is the number of mice used to determinethe values.

Mice treated with 2A4 showed decreased serum blood urea nitrogen (BUN)and alanine aminotransferase (ALT) levels when compared to mice treatedwith JH70. BUN and ALT are markers of renal and hepatic function,respectively, and their reduced levels indicate that organ function mayhave been better preserved by 2A4 treatment.

1. An antibody comprising a light chain variable region comprising theamino acid sequence set forth as SEQ ID NO:
 157. 2. An antibodycomprising a heavy chain variable region comprising the amino acidsequence set forth as SEQ ID NO:
 163. 3. An antibody comprising a lightchain variable region comprising the amino acid sequence set forth asSEQ ID NO: 157 and a heavy chain variable region comprising the aminoacid sequence set forth as SEQ ID NO:
 163. 4. The antibody of claim 3,which is a humanized version of murine monoclonal antibody 2A4 (ATCCAccession Number PTA-9662).
 5. A pharmaceutical composition comprising ahumanized antibody comprising a light chain variable region comprisingthe amino acid sequence set forth as SEQ ID NO: 157, a heavy chainvariable region comprising the amino acid sequence set forth as SEQ IDNO: 163, and a pharmaceutically acceptable carrier.
 6. Thepharmaceutical composition of claim 5, which is formulated forparenteral administration.
 7. An isolated host cell comprising a nucleicacid encoding the amino acid sequence set forth as SEQ ID NO:
 157. 8.The host cell of claim 7, which is a mammalian cell selected from thegroup consisting of CHO cells, HEK-293 cells, HeLa cells, CV-1 cells,and COS cells.
 9. The host cell of claim 7, wherein the host cell isexpressing an antibody light chain variable region comprising the aminoacid sequence set forth as SEQ ID NO:
 157. 10. An isolated host cellcomprising a nucleic acid encoding the amino acid sequence set forth asSEQ ID NO:
 163. 11. The host cell of claim 10, which is a mammalian cellselected from the group consisting of CHO cells, HEK-293 cells, HeLacells, CV-1 cells, and COS cells.
 12. The host cell of claim 10, whereinthe host cell is expressing an antibody heavy chain variable regioncomprising the amino acid sequence set forth as SEQ ID NO:
 163. 13. ACHO cell comprising at least one nucleic acid encoding the amino acidsequence set forth as SEQ ID NO: 157 and the amino acid sequence setforth as SEQ ID NO:
 163. 14. The CHO cell of claim 13, wherein the hostcell is expressing an antibody comprising a light chain variable regioncomprising the amino acid sequence set forth as SEQ ID NO: 157 and aheavy chain variable region comprising the amino acid sequence set forthas SEQ ID NO:
 163. 15. A recombinant antibody comprising a light chainvariable region comprising three complementarity determining regions setforth as SEQ ID NOs: 168, 169, and 170, and a heavy chain variableregion comprising three complementarity regions set forth as SEQ ID NOs:171, 172, and
 173. 16. The antibody of claim 15, wherein the antibody ismurine monoclonal antibody 2A4 (ATCC Accession Number PTA-9662).
 17. Theantibody of claim 15, which is a humanized version of murine monoclonalantibody 2A4 (ATCC Accession Number PTA-9662).
 18. A hybridomaexpressing murine monoclonal antibody 2A4 (ATCC Accession NumberPTA-9662).